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Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

In neuroinflammation, distinguishing microglia from macrophages and identifying microglial-specific biomarkers in peripheral blood pose significant challenges. This study comprehensively profiled the extracellular vesicles (EVs) of microglia and macrophages, respectively, revealing co-expressed EVs with UCHL1 and CX3CR1 as EVs derived specifically from microglia in human blood. After extensive validation, using optimized nano flow cytometry, we evaluated plasma CX3CR1+/UCHL1+ EVs across clinical cohorts [multiple sclerosis (MS), HTLV-1 associated myelopathy (HAM), Alzheimer's disease (AD), and Parkinson's disease (PD)], along with established neurodegenerative markers (NMDAR2A and NFL). The findings discovered a notable rise in CX3CR1+/UCHL1+ EVs in MS, particularly heightened in HAM, in contrast to controls. Conversely, AD and PD exhibited unaltered or diminished levels of microglial EVs. An integrated model of CX3CR1+/UCHL1+, NMDAR2A+, and NFL+ EVs demonstrated promising diagnostic potential for distinguishing MS from controls and HAM. As to the disease duration, CX3CR1+/UCHL1+ EVs increased in the initial five years of MS, stabilizing thereafter, whereas NMDAR2A+ and NFL+ EVs remained stable initially but increased significantly in the subsequent five years, suggesting their correlation with disease duration. This study uncovers unique blood microglial EVs with potential as biomarkers for MS diagnosis, differentiation from HAM, and correlation with disease duration. [ABSTRACT FROM AUTHOR]

Permanent inflammatory demyelinating and neurodegenerative processes lead to neurological disability in patients with multiple sclerosis (MS). The anti-inflammatory properties of vitamin D3 (VitD) are well established, but its role in neurodegeneration is still uncertain. The usefulness of the serum concentration of VitD as a potential biomarker in evaluating brain injury in terms of recently known smoldering MS was under consideration. Methods: We assessed the concentrations of the parameters of brain injury (NF-H, GPAF, S100B, UCHL1) in the cerebrospinal fluid (CSF) of relapsing-remitting (RRMS, n = 123) and progressive MS (PMS, n = 88) patients in the group with normal levels of VitD (VitDn) and in the VitD deficiency group (VitDd). The levels of NF-H and UCHL1 were higher in the group of VitDd compared to VitDn. The higher serum levels of VitD were correlated with lower concentrations of GFAP, NF-H and S100B in the CSF of the whole group of MS patients and in women with MS as opposed to the levels of UCHL1. In men, there were noted negative correlations between the levels of serum VitD and GFAP and NF-H in CSF but not between VitD and S100B and UCHL1. The negative correlations were observed between VitD and the selected parameters of brain injury in MS patients, in women as well as in men. The concentrations of serum VitD together with selected parameters of brain injury in CSF seem to be promising biomarkers of neurodegeneration processes in smoldering MS. [ABSTRACT FROM AUTHOR]

Background: Inflammatory demyelination and impaired recovery processes result in permanent neurodegeneration and neurological disability in patients with multiple sclerosis (MS). In terms of smoldering MS, chronic neuroinflammation develops in the early period of the disease and leads to confirmed disability accumulation. There is a great need to identify biomarkers of neurodegeneration and disease progression. Methods: A single-center prospective observational study was performed. The median age of the patients was 40 (31–52) years. Women comprised 64% of the study population. We evaluated the concentrations of the parameters of brain injury (NF-H, GFAP, S100B and UCHL1) in the cerebrospinal fluid (CSF) and the selected interleukins (ILs) in serum of 123 relapsing–remitting MS (RRMS) and 88 progressive MS (PMS) patients. Results: The levels of GFAP, S100B and UCHL were higher in the PMS group than the RRMS group, in contrast to the levels of NF-H. We observed a positive correlation between the selected pro-inflammatory cytokines and the parameters of brain injury. The Expanded Disability Status Scale (EDSS) score increased with GFAP and NF-H levels and was correlated with the selected ILs. The concentrations of S100B, UCHL1 and NF-H reflected the duration of MS symptoms. Conclusions: The levels of brain injury parameters in the CSF and the selected serum ILs in MS patients seem to be promising biomarkers to determine neurodegeneration and neuroinflammation in smoldering MS. Further studies are warranted in this respect. [ABSTRACT FROM AUTHOR]

Introduction: Ubiquitin C-terminal hydrolase L1 (UCHL1) has been associated with a severe, complex autosomal recessive spastic paraplegia (HSP79) [1] [2] [3] [4]. More recently, UCHL1 loss of function (LoF) variants have been associated to an autosomal dominant disease characterized by late-onset spastic ataxia, neuropathy, and frequent optic atrophy [5]. Methods: Routine clinical care whole-genome (WGS) and exome (ES) sequencing. Results: We present three families with autosomal dominant UCHL1-related disorder. The clinical phenotype mainly associated optic atrophy, mixed cerebellar and sensory ataxia, and possible hearing loss. We delineated two major phenotypes, even within the same family: (1) juvenile severe optic atrophy followed by a later-onset ataxia, or (2) late-onset ataxia with asymptomatic or mild optic atrophy. The families harboured three novel heterozygous variants in UCHL1: two loss of function (p.Lys115AsnfsTer40; c.171_174 + 7del11), and one missense (p.Asp176Asn) involving the catalytic site of the protein and potentially altering the adjacent splice site. Discussion: We confirm the existence of dominantly inherited UCHL1 pathogenic variants. We describe a considerable intrafamilial phenotypic variability, with two main phenotypes. Optic atrophy was consistently present, but with varying degrees of severity. Neither delayed motor or intellectual development, nor dysmorphic features were part of the dominant phenotype in comparison with the autosomal recessive form. The molecular mechanism appears to be haploinsufficiency. UCHL1 monoallelic variants should therefore be considered in any case of early-onset optic atrophy or in late-onset complex ataxic syndrome with asymptomatic optic atrophy. [ABSTRACT FROM AUTHOR]

Abstract In neuroinflammation, distinguishing microglia from macrophages and identifying microglial-specific biomarkers in peripheral blood pose significant challenges. This study comprehensively profiled the extracellular vesicles (EVs) of microglia and macrophages, respectively, revealing co-expressed EVs with UCHL1 and CX3CR1 as EVs derived specifically from microglia in human blood. After extensive validation, using optimized nano flow cytometry, we evaluated plasma CX3CR1+/UCHL1+ EVs across clinical cohorts [multiple sclerosis (MS), HTLV-1 associated myelopathy (HAM), Alzheimer’s disease (AD), and Parkinson’s disease (PD)], along with established neurodegenerative markers (NMDAR2A and NFL). The findings discovered a notable rise in CX3CR1+/UCHL1+ EVs in MS, particularly heightened in HAM, in contrast to controls. Conversely, AD and PD exhibited unaltered or diminished levels of microglial EVs. An integrated model of CX3CR1+/UCHL1+, NMDAR2A+, and NFL+ EVs demonstrated promising diagnostic potential for distinguishing MS from controls and HAM. As to the disease duration, CX3CR1+/UCHL1+ EVs increased in the initial five years of MS, stabilizing thereafter, whereas NMDAR2A+ and NFL+ EVs remained stable initially but increased significantly in the subsequent five years, suggesting their correlation with disease duration. This study uncovers unique blood microglial EVs with potential as biomarkers for MS diagnosis, differentiation from HAM, and correlation with disease duration.

Background Ubiquitin C-terminal hydrolase L1 (UCHL1), which encodes thiol protease that hydrolyzes a peptide bond at the C-terminal glycine residue of ubiquitin, regulates cell differentiation, proliferation, transcriptional regulation, and numerous other biological processes and may be involved in lung cancer progression. UCHL1 is mainly expressed in the brain and plays a tumor-promoting role in a few cancer types; however, there are limited reports regarding its role in lung cancer. Methods Single-cell RNA (scRNA) sequencing using 10X chromium v3 was performed on a paired normal-appearing and tumor tissue from surgical specimens of a patient who showed unusually rapid progression. To validate clinical implication of the identified biomarkers, immunohistochemical (IHC) analysis was performed on 48 non-small cell lung cancer (NSCLC) tissue specimens, and the correlation with clinical parameters was evaluated. Results We identified 500 genes overexpressed in tumor tissue compared to those in normal tissue. Among them, UCHL1, brain expressed X-linked 3 (BEX3), and midkine (MDK), which are associated with tumor growth and progression, exhibited a 1.5-fold increase in expression compared to that in normal tissue. IHC analysis of NSCLC tissues showed that only UCHL1 was specifically overexpressed. Additionally, in 48 NSCLC specimens, UCHL1 was specifically upregulated in the cytoplasm and nuclear membrane of tumor cells. Multivariable logistic analysis identified several factors, including smoking, tumor size, and high-grade dysplasia, to be typically associated with UCHL1 overexpression. Survival analyses using The Cancer Genome Atlas (TCGA) datasets revealed that UCHL1 overexpression is substantially associated with poor survival outcomes. Furthermore, a strong association was observed between UCHL1 expression and the clinicopathological features of patients with NSCLC. Conclusion UCHL1 overexpression was associated with smoking, tumor size, and high-grade dysplasia, which are typically associated with a poor prognosis and survival outcome. These findings suggest that UCHL1 may serve as an effective biomarker of NSCLC.

Abstract Background The proliferation of porcine ovarian granulosa cells (GCs) is essential to follicular development and the ubiquitin–proteasome system is necessary for maintaining cell cycle homeostasis. Previous studies found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) regulates female reproduction, especially in ovarian development. However, the mechanism by which UCHL1 regulates porcine GC proliferation remains unclear. Results UCHL1 overexpression promoted GC proliferation, and knockdown had the opposite effect. UCHL1 is directly bound to cyclin B1 (CCNB1), prolonging the half-life of CCNB1 and inhibiting its degradation, thereby promoting GC proliferation. What’s more, a flavonoid compound-isovitexin improved the enzyme activity of UCHL1 and promoted the proliferation of porcine GCs. Conclusions UCHL1 promoted the proliferation of porcine GCs by stabilizing CCNB1, and isovitexin enhanced the enzyme activity of UCHL1. These findings reveal the role of UCHL1 and the potential of isovitexin in regulating proliferation and provide insights into identifying molecular markers and nutrients that affect follicle development.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme originally found in the brain. Our previous work revealed that UCHL1 was also expressed in skeletal muscle and affected myoblast differentiation and metabolism. In this study, we further tested the role of UCHL1 in myogenesis and muscle regeneration following muscle ischemia-reperfusion (IR) injury. In the C2C12 myoblast, UCHL1 knockdown upregulated MyoD and myogenin and promoted myotube formation. The skeletal muscle-specific knockout (smKO) of UCHL1 increased muscle fiber sizes in young mice (1 to 2 months old) but not in adult mice (3 months old). In IR-injured hindlimb muscle, UCHL1 was upregulated. UCHL1 smKO ameliorated tissue damage and injury-induced inflammation. UCHL1 smKO also upregulated myogenic factors and promoted functional recovery in IR injury muscle. Moreover, UCHL1 smKO increased Akt and Pink1/Parkin activities. The overall results suggest that skeletal muscle UCHL1 is a negative factor in skeletal muscle development and recovery following IR injury and therefore is a potential therapeutic target to improve muscle regeneration and functional recovery following injuries. [ABSTRACT FROM AUTHOR]

Background: The proliferation of porcine ovarian granulosa cells (GCs) is essential to follicular development and the ubiquitin–proteasome system is necessary for maintaining cell cycle homeostasis. Previous studies found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) regulates female reproduction, especially in ovarian development. However, the mechanism by which UCHL1 regulates porcine GC proliferation remains unclear. Results: UCHL1 overexpression promoted GC proliferation, and knockdown had the opposite effect. UCHL1 is directly bound to cyclin B1 (CCNB1), prolonging the half-life of CCNB1 and inhibiting its degradation, thereby promoting GC proliferation. What's more, a flavonoid compound-isovitexin improved the enzyme activity of UCHL1 and promoted the proliferation of porcine GCs. Conclusions: UCHL1 promoted the proliferation of porcine GCs by stabilizing CCNB1, and isovitexin enhanced the enzyme activity of UCHL1. These findings reveal the role of UCHL1 and the potential of isovitexin in regulating proliferation and provide insights into identifying molecular markers and nutrients that affect follicle development. [ABSTRACT FROM AUTHOR]

Abstract Background Miscarriage is a frustrating complication of pregnancy that is common among women of reproductive age. Insufficient decidualization which not only impairs embryo implantation but disturbs fetomaternal immune-tolerance, has been widely regarded as a major cause of miscarriage; however, the underlying mechanisms resulting in decidual impairment are largely unknown. Methods With informed consent, decidual tissue from patients with spontaneous abortion or normal pregnant women was collected to detect the expression profile of UCHL1. Human endometrial stromal cells (HESCs) were used to explore the roles of UCHL1 in decidualization and dNK modulation, as well as the mechanisms involved. C57/BL6 female mice (7–10 weeks old) were used to construct pregnancy model or artificially induced decidualization model to evaluate the effect of UCHL1 on mice decidualization and pregnancy outcome. Results The Ubiquitin C-terminal hydrolase L1 (UCHL1), as a deubiquitinating enzyme, was significantly downregulated in decidua from patients with miscarriage, along with impaired decidualization and decreased dNKs. Blockage of UCHL1 led to insufficient decidualization and resultant decreased expression of cytokines CXCL12, IL-15, TGF-β which were critical for generation of decidual NK cells (dNKs), whereas UCHL1 overexpression enhanced decidualization accompanied by increase in dNKs. Mechanistically, the promotion of UCHL1 on decidualization was dependent on its deubiquitinating activity, and intervention of UCHL1 inhibited the activation of JAK2/STAT3 signaling pathway, resulting in aberrant decidualization and decreased production of cytokines associated with dNKs modulation. Furthermore, we found that inhibition of UCHL1 also disrupted the decidualization in mice and eventually caused adverse pregnancy outcome. Conclusions UCHL1 plays significant roles in decidualization and dNKs modulation during pregnancy in both humans and mice. Its deficiency indicates a poor pregnancy outcome due to defective decidualization, making UCHL1 a potential target for the diagnosis and treatment of miscarriage. Graphical Abstract

Abstract Background Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that regulates ERα expression in triple-negative cancer (TNBC). This study aimed to explore the deubiquitination substrates of UCHL1 related to endocrine therapeutic responses and the mechanisms of UCHL1 dysregulation in TNBC. Methods Bioinformatics analysis was conducted using online open databases. TNBC representative MDA-MB-468 and SUM149 cells were used for in vitro and in-vivo studies. Co-immunoprecipitation was used to explore the interaction between UCHL1 and KLF5 and UCHL1-mediated KIF5 deubiquitination. CCK-8, colony formation and animal studies were performed to assess endocrine therapy responses. The regulatory effect of TET1/3 on UCHL1 promoter methylation and transcription was performed by Bisulfite sequencing PCR and ChIP-qPCR. Results UCHL1 interacts with KLF5 and stabilizes KLF5 by reducing its polyubiquitination and proteasomal degradation. The UCHL1-KLF5 axis collaboratively upregulates EGFR expression while downregulating ESR1 expression at both mRNA and protein levels in TNBC. UCHL1 knockdown slows the proliferation of TNBC cells and sensitizes the tumor cells to Tamoxifen and Fulvestrant. KLF5 overexpression partially reverses these trends. Both TET1 and TET3 can bind to the UCHL1 promoter region, reducing methylation of associated CpG sites and enhancing UCHL1 transcription in TNBC cell lines. Additionally, TET1 and TET3 elevates KLF5 protein level in a UCHL1-dependent manner. Conclusion UCHL1 plays a pivotal role in TNBC by deubiquitinating and stabilizing KLF5, contributing to endocrine therapy resistance. TET1 and TET3 promote UCHL1 transcription through promoter demethylation and maintain KLF5 protein level in a UCHL1-dependent manner, implying their potential as therapeutic targets in TNBC.

Background: Miscarriage is a frustrating complication of pregnancy that is common among women of reproductive age. Insufficient decidualization which not only impairs embryo implantation but disturbs fetomaternal immune-tolerance, has been widely regarded as a major cause of miscarriage; however, the underlying mechanisms resulting in decidual impairment are largely unknown. Methods: With informed consent, decidual tissue from patients with spontaneous abortion or normal pregnant women was collected to detect the expression profile of UCHL1. Human endometrial stromal cells (HESCs) were used to explore the roles of UCHL1 in decidualization and dNK modulation, as well as the mechanisms involved. C57/BL6 female mice (7–10 weeks old) were used to construct pregnancy model or artificially induced decidualization model to evaluate the effect of UCHL1 on mice decidualization and pregnancy outcome. Results: The Ubiquitin C-terminal hydrolase L1 (UCHL1), as a deubiquitinating enzyme, was significantly downregulated in decidua from patients with miscarriage, along with impaired decidualization and decreased dNKs. Blockage of UCHL1 led to insufficient decidualization and resultant decreased expression of cytokines CXCL12, IL-15, TGF-β which were critical for generation of decidual NK cells (dNKs), whereas UCHL1 overexpression enhanced decidualization accompanied by increase in dNKs. Mechanistically, the promotion of UCHL1 on decidualization was dependent on its deubiquitinating activity, and intervention of UCHL1 inhibited the activation of JAK2/STAT3 signaling pathway, resulting in aberrant decidualization and decreased production of cytokines associated with dNKs modulation. Furthermore, we found that inhibition of UCHL1 also disrupted the decidualization in mice and eventually caused adverse pregnancy outcome. Conclusions: UCHL1 plays significant roles in decidualization and dNKs modulation during pregnancy in both humans and mice. Its deficiency indicates a poor pregnancy outcome due to defective decidualization, making UCHL1 a potential target for the diagnosis and treatment of miscarriage. [ABSTRACT FROM AUTHOR]

In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae may be a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. In this study, we investigated the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in the process of the transdifferentiation of auditory supporting cells (SCs) into hair cells (HCs). The expression of UCHL1 gradually decreased as HCs developed and was restricted to inner pillar cells and third-row Deiters' cells between P2 and P7, suggesting that UCHL1-expressing cells are similar to the cells with Lgr5-positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ-secretase inhibitor and Wnt agonist. Moreover, the inhibition of UCHL1 by LDN-57444 led to an increase in HC numbers. Mechanistically, LDN-57444 increased mTOR complex 1 activity and allowed SCs to transdifferentiate into HCs. The suppression of UCHL1 induces the transdifferentiation of auditory SCs and progenitors into HCs by regulating the mTOR pathway. [ABSTRACT FROM AUTHOR]

Background: Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that regulates ERα expression in triple-negative cancer (TNBC). This study aimed to explore the deubiquitination substrates of UCHL1 related to endocrine therapeutic responses and the mechanisms of UCHL1 dysregulation in TNBC. Methods: Bioinformatics analysis was conducted using online open databases. TNBC representative MDA-MB-468 and SUM149 cells were used for in vitro and in-vivo studies. Co-immunoprecipitation was used to explore the interaction between UCHL1 and KLF5 and UCHL1-mediated KIF5 deubiquitination. CCK-8, colony formation and animal studies were performed to assess endocrine therapy responses. The regulatory effect of TET1/3 on UCHL1 promoter methylation and transcription was performed by Bisulfite sequencing PCR and ChIP-qPCR. Results: UCHL1 interacts with KLF5 and stabilizes KLF5 by reducing its polyubiquitination and proteasomal degradation. The UCHL1-KLF5 axis collaboratively upregulates EGFR expression while downregulating ESR1 expression at both mRNA and protein levels in TNBC. UCHL1 knockdown slows the proliferation of TNBC cells and sensitizes the tumor cells to Tamoxifen and Fulvestrant. KLF5 overexpression partially reverses these trends. Both TET1 and TET3 can bind to the UCHL1 promoter region, reducing methylation of associated CpG sites and enhancing UCHL1 transcription in TNBC cell lines. Additionally, TET1 and TET3 elevates KLF5 protein level in a UCHL1-dependent manner. Conclusion: UCHL1 plays a pivotal role in TNBC by deubiquitinating and stabilizing KLF5, contributing to endocrine therapy resistance. TET1 and TET3 promote UCHL1 transcription through promoter demethylation and maintain KLF5 protein level in a UCHL1-dependent manner, implying their potential as therapeutic targets in TNBC. [ABSTRACT FROM AUTHOR]

Aim: X‐linked variants in Filamin A (FLNA) are associated with the Ehlers‐Danlos‐syndrome‐variant form of periventricular heterotopia, and autosomal dominant variants in ubiquitin C‐terminal hydrolase L1 (UCHL1) are associated with a late‐onset spastic ataxia, peripheral neuropathy and optic atrophy. Here we present a rare case involving both a novel heterozygous whole‐gene deletion of UCHL1 and a heterozygous frameshift variant in the FLNA gene resulting in a complex phenotype. Methods: A 67‐year‐old female with a confirmed pathogenic variant in the FLNA gene, resulting in an enlarged aorta and joint pains, presented with a 4‐year history of severe sensory ataxia, upper motor neuron signs, eye movement abnormalities and severe sensory loss. Results: Neurophysiology including Somatosensory‐evoked potentials confirmed the sensory loss as predominantly preganglionic with denervation. Genetic testing revealed a digenic cause of her complex presentation, confirming a pathogenic frameshift variant in the FLNA gene and a heterozygous loss of function deletion in the UCHL1 gene. Conclusions: To the best of our knowledge, this is the first case with concomitant pathogenic variants in the FLNA and UCHL1 genes which explain the complex phenotype. The severe preganglionic sensory loss is also a rare finding and expands the phenotype of UCHL1 variants. [ABSTRACT FROM AUTHOR]

Background: Bevacizumab resistance poses barriers to targeted therapy in clear cell renal cell carcinoma (ccRCC). Whether there exist epigenetic targets that modulate bevacizumab sensitivity in ccRCC remains indefinite. The focus of this study is to explore the role of UCHL1 in ccRCC. Methods: Both in vitro and in vivo experiments were utilized to investigate the roles of UCHL1 in ccRCC. In vivo ubiquitination assays were performed to validate the posttranslational modification of KDM4B by UCHL1. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to explore KDM4B/VEGFA epigenetic regulations. Results: UCHL1 was increased in ccRCC and associated with unfavorable survival outcomes in patients. UCHL1 was required for ccRCC growth and migration. Mechanistically, the wild-type UCHL1, but not C90A mutant, mediated the deubiquitination of KDM4B and thereby stabilized its proteins. KDM4B was up-regulated in ccRCC and potentiated cell growth. UCHL1 depended on KDM4B to augment ccRCC malignancies. Targeting UCHL1 suppressed tumor growth, colony formation, and migration abilities, which could be rescued by KDM4B. Furthermore, KDM4B was directly bound to the promoter region of VEGFA, abolishing repressive H3K9me3 modifications. KDM4B coordinated with HIF2α to activate VEGFA transcriptional levels. UCHL1-KDM4B axis governs VEGFA levels to sustain the angiogenesis phenotypes. Finally, a specific small-molecule inhibitor (6RK73) targeting UCHL1 remarkably inhibited ccRCC progression and further sensitized ccRCC to bevacizumab treatment. Conclusion: Overall, this study defined an epigenetic mechanism of UCHL1/KDM4B in activating VEGF signaling. The UCHL1-KDM4B axis represents a novel target for treating ccRCC and improving the efficacy of anti-angiogenesis therapy.

Permanent inflammatory demyelinating and neurodegenerative processes lead to neurological disability in patients with multiple sclerosis (MS). The anti-inflammatory properties of vitamin D3 (VitD) are well established, but its role in neurodegeneration is still uncertain. The usefulness of the serum concentration of VitD as a potential biomarker in evaluating brain injury in terms of recently known smoldering MS was under consideration. Methods: We assessed the concentrations of the parameters of brain injury (NF-H, GPAF, S100B, UCHL1) in the cerebrospinal fluid (CSF) of relapsing-remitting (RRMS, n = 123) and progressive MS (PMS, n = 88) patients in the group with normal levels of VitD (VitDn) and in the VitD deficiency group (VitDd). The levels of NF-H and UCHL1 were higher in the group of VitDd compared to VitDn. The higher serum levels of VitD were correlated with lower concentrations of GFAP, NF-H and S100B in the CSF of the whole group of MS patients and in women with MS as opposed to the levels of UCHL1. In men, there were noted negative correlations between the levels of serum VitD and GFAP and NF-H in CSF but not between VitD and S100B and UCHL1. The negative correlations were observed between VitD and the selected parameters of brain injury in MS patients, in women as well as in men. The concentrations of serum VitD together with selected parameters of brain injury in CSF seem to be promising biomarkers of neurodegeneration processes in smoldering MS.

Background: Inflammatory demyelination and impaired recovery processes result in permanent neurodegeneration and neurological disability in patients with multiple sclerosis (MS). In terms of smoldering MS, chronic neuroinflammation develops in the early period of the disease and leads to confirmed disability accumulation. There is a great need to identify biomarkers of neurodegeneration and disease progression. Methods: A single-center prospective observational study was performed. The median age of the patients was 40 (31–52) years. Women comprised 64% of the study population. We evaluated the concentrations of the parameters of brain injury (NF-H, GFAP, S100B and UCHL1) in the cerebrospinal fluid (CSF) and the selected interleukins (ILs) in serum of 123 relapsing–remitting MS (RRMS) and 88 progressive MS (PMS) patients. Results: The levels of GFAP, S100B and UCHL were higher in the PMS group than the RRMS group, in contrast to the levels of NF-H. We observed a positive correlation between the selected pro-inflammatory cytokines and the parameters of brain injury. The Expanded Disability Status Scale (EDSS) score increased with GFAP and NF-H levels and was correlated with the selected ILs. The concentrations of S100B, UCHL1 and NF-H reflected the duration of MS symptoms. Conclusions: The levels of brain injury parameters in the CSF and the selected serum ILs in MS patients seem to be promising biomarkers to determine neurodegeneration and neuroinflammation in smoldering MS. Further studies are warranted in this respect.

Improving the function of the blood–spinal cord barrier (BSCB) benefits the functional recovery of mice following spinal cord injury (SCI). The death of endothelial cells and disruption of the BSCB at the injury site contribute to secondary damage, and the ubiquitin–proteasome system is involved in regulating protein function. However, little is known about the regulation of deubiquitinated enzymes in endothelial cells and their effect on BSCB function after SCI. We observed that Sox17 is predominantly localized in endothelial cells and is significantly upregulated after SCI and in LPS-treated brain microvascular endothelial cells. In vitro Sox17 knockdown attenuated endothelial cell proliferation, migration, and tube formation, while in vivo Sox17 knockdown inhibited endothelial regeneration and barrier recovery, leading to poor functional recovery after SCI. Conversely, in vivo overexpression of Sox17 promoted angiogenesis and functional recovery after injury. Additionally, immunoprecipitation-mass spectrometry revealed the interaction between the deubiquitinase UCHL1 and Sox17, which stabilized Sox17 and influenced angiogenesis and BSCB repair following injury. By generating UCHL1 conditional knockout mice and conducting rescue experiments, we further validated that the deubiquitinase UCHL1 promotes angiogenesis and restoration of BSCB function after injury by stabilizing Sox17. Collectively, our findings present a novel therapeutic target for treating SCI by revealing a potential mechanism for endothelial cell regeneration and BSCB repair after SCI. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) play a crucial role in mammalian spermatogenesis and maintain the stable inheritance of the germline in livestock. However, stress and bacterial or viral infections can disrupt immune homeostasis of the testes, thereby leading to spermatogenesis destruction and infertility, which severely affects the health and productivity of mammals. This study aimed to explore the effect of ubiquitin C-terminal hydrolase L1 (UCHL1) knockdown (KD) in goat SSCs and mouse testes and investigate the potential anti-inflammatory function of UCHL1 in a poly(I:C)-induced inflammation model to maintain microenvironmental homeostasis. In vitro, the downregulation of UCHL1 (UCHL1 KD) in goat SSCs increased the expression levels of apoptosis and inflammatory factors and inhibited the self-renewal and proliferation of SSCs. In vivo, the structure of seminiferous tubules and spermatogenic cells was disrupted after UCHL1 KD, and the expression levels of apoptosis- and inflammation-related proteins were significantly upregulated. Furthermore, UCHL1 inhibited the TLR3/TBK1/ IRF3 pathway to resist poly(I:C)-induced inflammation in SSCs by antagonizing HSPA8 and thus maintaining SSC autoimmune homeostasis. Most importantly, the results of this study showed that UCHL1 maintained immune homeostasis of SSCs and spermatogenesis. UCHL1 KD not only inhibited the self-renewal and proliferation of goat SSCs and spermatogenesis but was also involved in the inflammatory response of goat SSCs. Additionally, UCHL1 has an antiviral function in SSCs by antagonizing HSPA8, which provides an important basis for exploring the specific mechanisms of UCHL1 in goat spermatogenesis. [ABSTRACT FROM AUTHOR]

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme originally found in the brain. Our previous work revealed that UCHL1 was also expressed in skeletal muscle and affected myoblast differentiation and metabolism. In this study, we further tested the role of UCHL1 in myogenesis and muscle regeneration following muscle ischemia-reperfusion (IR) injury. In the C2C12 myoblast, UCHL1 knockdown upregulated MyoD and myogenin and promoted myotube formation. The skeletal muscle-specific knockout (smKO) of UCHL1 increased muscle fiber sizes in young mice (1 to 2 months old) but not in adult mice (3 months old). In IR-injured hindlimb muscle, UCHL1 was upregulated. UCHL1 smKO ameliorated tissue damage and injury-induced inflammation. UCHL1 smKO also upregulated myogenic factors and promoted functional recovery in IR injury muscle. Moreover, UCHL1 smKO increased Akt and Pink1/Parkin activities. The overall results suggest that skeletal muscle UCHL1 is a negative factor in skeletal muscle development and recovery following IR injury and therefore is a potential therapeutic target to improve muscle regeneration and functional recovery following injuries.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-β1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

Background: Multiple sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS). Due to the different phenotypes of the disease and non-specific symptoms of MS, there is a great need for a validated panel of biomarkers to facilitate the diagnosis, predict disease progression, and evaluate treatment outcomes. Methods: We determined the levels of the parameters of brain injury (NF-H, GPAF, S100B, and UCHL1) and the selected cytokines in the cerebrospinal fluid (CSF) in 101 patients diagnosed de novo with RRMS and 75 healthy controls. All determinations were made using the Bio-Plex method. Results: We found higher levels of NF-H and GFAP in the relapsing-remitting multiple sclerosis (RRMS) group compared to the controls. The concentrations of both molecules were significantly increased in patients with Gd+ lesions on brain MRI. The level of S100B did not differ significantly between the groups. UCHL1 concentrations were higher in the control group. We found some correlations between the selected cytokines, the levels of the parameters of brain injury, and the time from the first symptoms to the diagnosis of MS. Conclusions: The role of the above molecules in MS is promising. However, further research is warranted to define their precise functions. [ABSTRACT FROM AUTHOR]

The regulation of protein turnover by the unique deubiquitinating enzyme ubiquitin C-terminal hydrolase L1 (UCHL1) is only seen in oocytes, spermatogonia, and neurons. Our objective was to investigate variation in expression of UCHL1 across fetal maturation of oocytes that result in lifelong ovarian reserve. We performed a retrospective cohort study of 25 fetal autopsy specimens from 21 to 36 weeks. This was an IRB-approved protocol with parental permission for use of tissues for research purposes. Tissues were stained for expression of the oocyte-specific protein UCHL1, and expression levels were evaluated using quantitative immunofluorescence across gestational ages after correction for the area and background absorbance. Corrected total cell fluorescence (CTCF) for expression of UCHL1 within human oocytes was compared across fetal gestational ages and oocyte size. Trends were analyzed using a locally weighted scatterplot smoothing algorithm. Local expression of UCHL1 increases in oocytes across ovarian development reaching a plateau at 27 weeks with the maintenance of elevated levels through 36 weeks gestational age. This maturation trend is also evidenced by the increase in protein expression as oocyte area increases (r = 0.5530, p ≤ 0.001) with the largest rise occurring as oocytes are enveloped into primordial follicles. The increase in expression as oocytes transition from oogonia into oocytes in primordial follicles and beyond may be part of the preparation of both oocytes and the surrounding somatic cells for the long-term maintenance of the ovarian reserve. [ABSTRACT FROM AUTHOR]

GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a−/− mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a−/− mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1−/− (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy. [ABSTRACT FROM AUTHOR]

In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae may be a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. In this study, we investigated the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in the process of the transdifferentiation of auditory supporting cells (SCs) into hair cells (HCs). The expression of UCHL1 gradually decreased as HCs developed and was restricted to inner pillar cells and third-row Deiters’ cells between P2 and P7, suggesting that UCHL1-expressing cells are similar to the cells with Lgr5-positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ-secretase inhibitor and Wnt agonist. Moreover, the inhibition of UCHL1 by LDN-57444 led to an increase in HC numbers. Mechanistically, LDN-57444 increased mTOR complex 1 activity and allowed SCs to transdifferentiate into HCs. The suppression of UCHL1 induces the transdifferentiation of auditory SCs and progenitors into HCs by regulating the mTOR pathway.

Objectives: Traumatic brain injury (TBI) is one of the top causes of morbidity and mortality worldwide. The review aimed to discuss and summarize the current evidence on the effectiveness of adjuvant neuroprotective treatments in terms of their effect on brain injury biomarkers in TBI patients. Methods: To identify relevant studies, four scholarly databases, including PubMed, Cochrane, Scopus, and Google Scholar, were systematically searched using predefined search terms. English-language randomized controlled clinical trials reporting changes in brain injury biomarkers, namely, neuron-specific enolase (NSE), glial fibrillary acid protein (GFAP), ubiquitin carboxyl-terminal esterase L1 (UCHL1) and/or S100 beta (S100 ß), were included. The methodological quality of the included studies was assessed using the Cochrane risk-of-bias tool. Results: A total of eleven studies with eight different therapeutic options were investigated; of them, tetracyclines, metformin, and memantine were discovered to be promising choices that could improve neurological outcomes in TBI patients. The most utilized serum biomarkers were NSE and S100 ß followed by GFAP, while none of the included studies quantified UCHL1. The heterogeneity in injury severity categories and measurement timing may affect the overall evaluation of the clinical efficacy of potential therapies. Therefore, unified measurement protocols are highly warranted to inform clinical decisions. Conclusion: Few therapeutic options showed promising results as an adjuvant to standard care in patients with TBI. Several considerations for future work must be directed towards standardizing monitoring biomarkers. Investigating the pharmacotherapy effectiveness using a multimodal biomarker panel is needed. Finally, employing stratified randomization in future clinical trials concerning potential confounders, including age, trauma severity levels, and type, is crucial to inform clinical decisions. [ABSTRACT FROM AUTHOR]

Objectives: Traumatic brain injury (TBI) is one of the top causes of morbidity and mortality worldwide. The review aimed to discuss and summarize the current evidence on the effectiveness of adjuvant neuroprotective treatments in terms of their effect on brain injury biomarkers in TBI patients.Methods: To identify relevant studies, four scholarly databases, including PubMed, Cochrane, Scopus, and Google Scholar, were systematically searched using predefined search terms. English-language randomized controlled clinical trials reporting changes in brain injury biomarkers, namely, neuron-specific enolase (NSE), glial fibrillary acid protein (GFAP), ubiquitin carboxyl-terminal esterase L1 (UCHL1) and/or S100 beta (S100 ß), were included. The methodological quality of the included studies was assessed using the Cochrane risk-of-bias tool.Results: A total of eleven studies with eight different therapeutic options were investigated; of them, tetracyclines, metformin, and memantine were discovered to be promising choices that could improve neurological outcomes in TBI patients. The most utilized serum biomarkers were NSE and S100 ß followed by GFAP, while none of the included studies quantified UCHL1. The heterogeneity in injury severity categories and measurement timing may affect the overall evaluation of the clinical efficacy of potential therapies. Therefore, unified measurement protocols are highly warranted to inform clinical decisions.Conclusion: Few therapeutic options showed promising results as an adjuvant to standard care in patients with TBI. Several considerations for future work must be directed towards standardizing monitoring biomarkers. Investigating the pharmacotherapy effectiveness using a multimodal biomarker panel is needed. Finally, employing stratified randomization in future clinical trials concerning potential confounders, including age, trauma severity levels, and type, is crucial to inform clinical decisions.Clinical Trial Registration: [https://www.crd.york.ac.uk/prospero/dis], identifier [CRD42022316327].

Uterine leiomyomas are smooth-muscle tumors originating in the myometrium and are the most common pelvic tumors in women of reproductive age. Symptomatic tumors may result in abnormal uterine bleeding, bladder dysfunction, pelvic discomfort, and reproductive issues, such as infertility and miscarriage. There are currently few non-invasive treatments for leiomyoma, but there are no practical early intervention or preventive methods. In this study, human uterine leiomyoma and myometrial tissues were used to detect the protein and mRNA expression levels of UCHL1. To explore the effects of UCHL1 knockdown and inhibition in leiomyoma and myometrial cells, we determined the mRNA expressions of COL1A1 and COL3A1. Collagen gel contraction and wound-healing assays were performed on myometrial and leiomyoma cells. We found that UCHL1 expression was considerably higher in uterine leiomyomas than in the myometrium. COL1A1 and COL3A1 expression levels were downregulated after inhibition of UCHL1 in human leiomyoma cells. Furthermore, the elimination of UCHL1 significantly decreased the migration and contractility of leiomyoma cells. In conclusion, these results indicate that UCHL1 is involved in the growth of leiomyoma in humans. For the treatment of uterine leiomyoma, targeting UCHL1 activity may be a unique and possible therapeutic strategy. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS). Due to the different phenotypes of the disease and non-specific symptoms of MS, there is a great need for a validated panel of biomarkers to facilitate the diagnosis, predict disease progression, and evaluate treatment outcomes. Methods: We determined the levels of the parameters of brain injury (NF-H, GPAF, S100B, and UCHL1) and the selected cytokines in the cerebrospinal fluid (CSF) in 101 patients diagnosed de novo with RRMS and 75 healthy controls. All determinations were made using the Bio-Plex method. Results: We found higher levels of NF-H and GFAP in the relapsing-remitting multiple sclerosis (RRMS) group compared to the controls. The concentrations of both molecules were significantly increased in patients with Gd+ lesions on brain MRI. The level of S100B did not differ significantly between the groups. UCHL1 concentrations were higher in the control group. We found some correlations between the selected cytokines, the levels of the parameters of brain injury, and the time from the first symptoms to the diagnosis of MS. Conclusions: The role of the above molecules in MS is promising. However, further research is warranted to define their precise functions.

GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a−/− mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a−/− mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1−/− (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy.

ABSTRACT: A new undesirable genetic factor, neuropathy with splayed forelimbs (JNS), has been identified recently in the Jersey breed. Calves affected with JNS are unable to stand on splayed forelimbs that exhibit significant extensor rigidity and excessive lateral abduction at birth. Affected calves generally are alert at birth but exhibit neurologic symptoms, including spasticity of head and neck and convulsive behavior. Other symptoms reported include dislocated shoulders, congenital craniofacial anomalies, and degenerative myelopathy. Inheritance of an undesirable genetic factor was determined from a study of 16 affected calves reported by Jersey breeders across the United States. All of their pedigrees traced back on both paternal and maternal sides to a common ancestor born in 1995. Genotypes revealed that JNS is attributable to a specific haplotype on Bos taurus autosome 6. Currently 8.2% of the genotyped US Jersey population are carriers of the haplotype. Sequencing of the region of shared homozygosity revealed missense variant rs1116058914 at base 60,158,901 of the ARS-UCD1.2 reference map as the most concordant with the genetic condition and the most likely cause. The single-base G to A substitution is in the coding region of the last exon of UCHL1, which is conserved across species. Mutations in humans and gene knockouts in mice cause similar recessive symptoms and muscular degeneration. Since December 2020, carrier status has been tracked using the identified haplotype and reported for all 459,784 genotyped Jersey animals. With random mating, about 2,200 affected calves per year with losses of about $250,000 would result from the 1.3 million US Jersey cows in the national population. Selection and mating programs can reduce numbers of JNS-affected births using either the haplotype status or a direct gene test in the future. Breeders should report calf abnormalities to their breed association to help discover new defects such as JNS.

Yulan Huang,* Shitong He,* Yitian Chen, Junli Sheng, Yuling Fu, Xialin Du, Yalong Yang, Honglin Liu, Zhenyu Han, Yingqi Huang, Qian Wen, Chaoying Zhou, Xinying Zhou, Shengfeng Hu, Li Ma Institute of Molecular Immunology, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li Ma; Shengfeng HuInstitute of Molecular Immunology, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, People’s Republic of China, Email mali_61648322@smu.edu.cn; hushengfeng@smu.edu.cnBackground: As deubiquitinases (DUBs), ubiquitin C-terminal hydrolase (UCH)-L1 has been shown to play a crucial role in regulating diverse biological processes. However, its function in macrophage polarization remains unclear.Methods: We performed in vivo and in vitro experiments to investigate the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a kind of DUBs, in macrophage differentiation by using UCHL1-deficiency mice.Results: We demonstrated that LPS stimulation induced UCHL1 expression in macrophages. The deficiency of UCHL1 expression decreased the expression of CD80 and CD86 but increased the expression of CD206. The expression of TNF-α, IL-6, iNOS, and IL-10 was downregulated, while that of Arg1, Ym1, and Fizz1 was upregulated in UCHL1 deficient macrophages. Moreover, we observed that UCHL1 promoted the degradation of p110α through autophagy, but paradoxically increased the activity of AKT, thereby promoting polarization of macrophages into pro-inflammatory states.Conclusion: In this study, we identified UCHL1 as a positive regulator of M1 macrophage polarization. Our findings may help in developing therapeutic interventions for the treatment of inflammatory diseases and pathogenic infections.Keywords: UCHL1, macrophages, AKT, p110α, autophagy

Objective: To study the prevalence of spermatogonia in adult subjects with Klinefelter syndrome (KS) using MAGE-A4 and UCHL1 (PGP9.5) immunohistochemistry as markers for undifferentiated spermatogonial cells. We aimed to compare this method to the gold standard of hematoxylin and eosin (H & E) staining with histologic analysis in the largest reported cohort of adult subjects with KS.Design: A retrospective cohort study.Setting: Infertility Clinic and Institute for Regenerative Medicine.Patient(s): This study consisted of 79 adult subjects with KS and 12 adult control subjects.Intervention(s): The subjects with KS (n = 79) underwent bilateral testicular biopsy in an initial effort to recover spermatozoa for in vitro fertilization and intracytoplasmic sperm injection. The institutional review board approved the use of a portion of the archived diagnostic pathology paraffin blocks for the study. The samples were superimposed onto microscopic slides and labeled with the PGP9.5 and MAGE-A4 antibodies. Subjects (n = 12) who had previously consented to be organ donors via the National Disease Research Interchange were selected as controls. Dedicated genitourinary pathologists examined the H & E-, PGP9.5-, and MAGE-A4-stained tissue for presence of undifferentiated spermatogonia and spermatozoa with the use of a virtual microscopy software.Main Outcome Measure(s): The primary outcome was the presence of MAGE-A4-positive or UCHL1-positive tubules that indicate undifferentiated spermatogonia. Supportive outcomes include assessing the biopsy specimen for the following: total surface area; total seminiferous tubule surface area; total interstitium surface area; the total number of seminiferous tubules; and MAGE-A4- negative or UCHL1-negative tubules. Additionally, clinical information, such as age, karyotype, height, weight, mean testicle size, and hormonal panel (luteinizing hormone, follicle-stimulating hormone, and testosterone), was obtained and used in a single and multivariable analysis with linear regression to determine predictive factors for the number of UCHL1-positive tubules.Result(s): The mean age of the subjects in the KS group was 32.9 ± 0.7 years (range, 16-48). UCHL1 (PGP9.5) and MAGE-A4 staining showed that 74.7% (n = 59) and 40.5% (n = 32) of the subjects with KS, respectively, were positive for undifferentiated spermatogonia compared with 100% (n = 12) of the control subjects who were positive for both the markers. Hematoxylin and eosin with microscopic analysis showed that only 10.1% (n = 8) of the subjects were positive for spermatogonia. The mean number of positive tubules per subject with KS was 11.8 ± 1.8 for UCHL1 and 3.7 ± 1.0 for MAGE-A4. Secondary analysis showed 7 (8.9%) adult subjects with KS as positive for spermatozoa on biopsy. The population having negative testicular sperm extraction results (n = 72) showed a spermatogonia-positive rate of 1.4%, (n = 1), 72.2% (n = 52), and 34.7% (n = 25) using H & E, UCHL1, and MAGE-A4, respectively. Further analysis showed that 54 (75.0%) subjects were either positive for UCHL1 or MAGE-A4. Twenty (27.8%) subjects were positive for both UCHL1 and MAGE-A4. Multivariate analysis with linear regression showed no significant correlation between clinical variables and the number of UCHL1-positive tubules found on biopsy specimens.Conclusion(s): We report a cohort of adult subjects with KS undergoing analysis for the presence of undifferentiated spermatogonia. UCHL1 and MAGE-A4 immunostaining appear to be an effective way of identifying undifferentiated spermatogonia in testicular biopsy specimens of subjects with KS. Despite observing deterioration in the testicular architecture, many patients remain positive for undifferentiated spermatogonia, which could be harvested and potentially used for infertility therapy in a patient with KS who is azoospermic and has negative testicular sperm extraction results. [ABSTRACT FROM AUTHOR]

Ethnopharmacological Relevance: Uncaria rhynchophylla (Miq.) Miq. ex Havil. (UR), a traditional Chinese medicinal plant, plays an active role in neuroprotection. Clinical medication and modern pharmacological studies have proved the efficacy of UR against Parkinson's disease (PD), with alkaloids being recognized as the main bioactive components. But the therapeutic effect and mechanism of U. rhynchophylla alkaloid extract (URA) against PD need further exploration., Aim of Study: The study aimed to investigate the therapeutic effect and potential mechanism of URA on PD., Materials and Methods: LC-MS methodology was used to evaluate the chemical constituents of URA. The anti-PD activity of URA in vivo was measured on the mouse and rat models of PD. Neuroprotective effect of URA on PC12 cells was measured by MTT assay. Dopamine (DA) and its metabolites were detected by LC-MS for probing the protection ability on dopaminergic neurons. The differentially expressed proteins between model group and URA administrated group were analyzed by proteomics, suggesting oxidative phosphorylation as possible pathway of URA. Considering the critical role of mitochondria in oxidative phosphorylation, JC-1 staining, MitoSOX staining, transmission electron microscopy (TEM) observation and adenosine triphosphate (ATP) levels detection were used to analyze the effects of URA on mitochondrial morphology and function. Biolayer interferometry (BLI) was used to search for the possible UCHL1-bonding compounds in URA., Results: URA significantly mitigated the behavioural defects by improving coordination, shortening the time to climb down the whole pole (T-LA) and increasing the forelimbs' muscle strength of MPTP-induced PD mice and 6-OHDA-induced PD rats. In addition, URA improved tyrosine hydroxylase expression in dopaminergic neurons by immunohistochemistry (IHC) staining, and thus increased the neurotransmitter levels of DA and relevant metabolites. Furthermore, URA promoted mitophagy as reflected by a significant decrease in reactive oxygen species (ROS) generation, an increase in ATP levels and clearance of damaged mitochondria. Subsequently, Ubiquitin C-terminal hydrolase 1 (UCHL1), which is associated with the mitochondrial dysfunction in PD, is suggested to be a promising target based on the proteomics result, and proved by the blocked protective effects of URA by UCHL1 inhibitor. Furthermore, hirsuteine (HTE) was identified as a potential active compound of URA binding to UCHL1 by BLI, and the binding capacity and site were verified by surface plasmon resonance (SPR) and molecular docking., Conclusion: This work demonstrates that URA exerts effective neuroprotective activity against PD via activation of mitophagy with the involvement of UCHL1, and HTE may be a potential active compound of URA., Competing Interests: Declaration of competing interest The author is not an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for Journal of Ethnopharmacology and was not involved in the editorial review or the decision to publish this article. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: We state that there is no commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Ubiquitin C-terminal hydrolase L1 (UCHL1), which encodes thiol protease that hydrolyzes a peptide bond at the C-terminal glycine residue of ubiquitin, regulates cell differentiation, proliferation, transcriptional regulation, and numerous other biological processes and may be involved in lung cancer progression. UCHL1 is mainly expressed in the brain and plays a tumor-promoting role in a few cancer types; however, there are limited reports regarding its role in lung cancer., Methods: Single-cell RNA (scRNA) sequencing using 10X chromium v3 was performed on a paired normal-appearing and tumor tissue from surgical specimens of a patient who showed unusually rapid progression. To validate clinical implication of the identified biomarkers, immunohistochemical (IHC) analysis was performed on 48 non-small cell lung cancer (NSCLC) tissue specimens, and the correlation with clinical parameters was evaluated., Results: We identified 500 genes overexpressed in tumor tissue compared to those in normal tissue. Among them, UCHL1, brain expressed X-linked 3 (BEX3), and midkine (MDK), which are associated with tumor growth and progression, exhibited a 1.5-fold increase in expression compared to that in normal tissue. IHC analysis of NSCLC tissues showed that only UCHL1 was specifically overexpressed. Additionally, in 48 NSCLC specimens, UCHL1 was specifically upregulated in the cytoplasm and nuclear membrane of tumor cells. Multivariable logistic analysis identified several factors, including smoking, tumor size, and high-grade dysplasia, to be typically associated with UCHL1 overexpression. Survival analyses using The Cancer Genome Atlas (TCGA) datasets revealed that UCHL1 overexpression is substantially associated with poor survival outcomes. Furthermore, a strong association was observed between UCHL1 expression and the clinicopathological features of patients with NSCLC., Conclusion: UCHL1 overexpression was associated with smoking, tumor size, and high-grade dysplasia, which are typically associated with a poor prognosis and survival outcome. These findings suggest that UCHL1 may serve as an effective biomarker of NSCLC.

Ubiquitin is a small protein that is conjugated to target proteins to signal a great number of critical biological processes. Impaired ubiquitin signaling and defects in the ubiquitin proteasome system (UPS) surveillance are implicated in many human diseases, including cancer. Characterization of the physiological roles of UPS components and their regulatory mechanisms is therefore vital for the identification of therapeutic targets and the development of tools and paradigms to better understand and treat human diseases. In this Special Issue, we assembled seven original research and review articles to provide insights on the multifaceted role of the UPS in pathogenesis and disease, covering the areas of molecular and cellular mechanisms of UPS enzymes, biochemical and biophysical characterization strategies, drug development, and targeted protein degradation. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial dysfunction plays a crucial role in Parkinson’s disease (PD) pathogenesis. The present study was undertaken to investigate the effects of Telmisartan (TEL), an angiotensin II type 1 receptor (AT1R) blocker, on the mitochondria-specific genes expression in a mouse model of Parkinsonism. Materials and methods: Mice were divided into 5 groups with 6 in each; Group I received 0.5% CMC (control) + saline, Group II received 0.5% CMC + 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (positive control), Group III & IV received MPTP + TEL 3 and 10 mg/kg, p.o. respectively, Group V received TEL 10 mg/kg, p.o. (drug control). MPTP was given 80 mg/kg intraperitoneal in two divided doses (40 mg/kg × 2 at 16 h time interval). Vehicle or TEL was administered 1 h before the MPTP injection. Motor function was assessed 48 h after the first dose of MPTP and animals were euthanized to collect brain. Results: Mice intoxicated with MPTP showed locomotor deficits and significant upregulation of α-synuclein (α-syn), downregulation of metastasis-associated protein 1 (MTA1), and Ubiquitin C-terminal hydrolase L1 (UCHL1) in the substantia nigra pars compacta (SNpc) and Striatum (STr) regions of brains. In addition, MPTP intoxication down-regulated mitochondria-specific genes such as DJ-1, PTEN-induced putative kinase 1 (PINK1), Parkin, enriched with leucine repeats kinase 2 (LRRK2) gene expfression. Pre-treatment with TEL restored locomotor functions and upregulated PINK1, Parkin, LRRK2, DJ-1, MTA1 and UCHL1. Conclusion: The present study evidences that TEL has the ability to improve mitochondrial functions in PD.

A cavernous hemangioma, well-known as vascular malformation, is present at birth, grows proportionately with the child, and does not undergo regression. Although a cavernous hemangioma has well-defined histopathological characteristics, its origin remains controversial. In the present study, we characterized the cellular heterogeneity of a cavernous hemangioma using single-cell RNA sequencing (scRNA-seq). The main contribution of the present study is that we discovered a large number of embryonic mesenchymal stem cells (MSCs) in a cavernous hemangioma and proposed that cavernous hemangiomas may originate from embryonic MSCs. Further analysis of the embryonic MSCs revealed that: 1) proinflammatory cytokines and related genes TNF, TNFSF13B, TNFRSF12A, TNFAIP6, and C1QTNF6 are significantly involved in the MSC-induced immune responses in cavernous hemangiomas; 2) UCHL1 is up-regulated in the embryonic MSC apoptosis induced by proinflammatory cytokines; 3) the UCHL1-induced apoptosis of MSCs may play an important role in the MSC-induced immune responses in cavernous hemangiomas; and 4) UCHL1 can be used as a marker gene to detect embryonic MSCs at different apoptosis stages. In addition to MSCs, ECs, macrophages, T lymphocytes and NKCs were intensively investigated, revealing the genes and pathways featured in cavernous hemangiomas. The present study revealed the origin of cavernous hemangiomas and reported the marker genes, cell types and molecular mechanisms, which are associated with the origin, formation, progression, diagnosis and therapy of cavernous hemangiomas. The better understanding of the MSC-induced immune responses in benign tumours helps to guide future investigation and treatment of embryonic MSC-caused tumours. Our findings initiated future research for the rediscovery of MSCs, cancers/tumours and the UCHL1-induced apoptosis.

Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1−/− hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1−/− cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1−/− cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinase known to play essential roles in the nervous tissue. Myocardial upregulation of UCHL1 was observed in human dilated cardiomyopathy and several animal models of heart disease, but the (patho)physiological significance of UCHL1 in cardiomyocytes remains undefined. Hence, we conducted this study to fill this critical gap. We produced cardiomyocyte-restricted Uchl1 knockout (CKO) by coupling the Uchl1-floxed allele with transgenic Myh6-Cre in C57B/6J inbred mice. Mice transgenic for Myh6-Cre were used as controls (CTL). Myocardial Uchl1 proteins were markedly reduced in CKO mice but they did not display discernible abnormal phenotype. Ten-week old CTL or CKO mice were subjected to left anterior descending artery ligation (myocardial infarction, MI) or sham surgery (Sham) and characterized at 7- and 28-day after surgery. Compared with Sham mice, significant increases in myocardial UCHL1 proteins were detected in CTL MI but not in CKO MI mice. MI-induced left ventricular (LV) chamber dilation, reduction of ejection fraction (EF) and fractional shortening (FS), and LV anterior wall thinning detected by echocardiography were comparable between the CTL MI and CKO MI groups 7-day post-MI. However, by 28-day post-MI, MI-induced LV chamber dilatation, EF and FS reduction, increases of myocardial ubiquitin conjugates, and increases in the heart weight to body weight ratio and the ventricular weight to body weight ratio were significantly more pronounced in CKO MI than CTL MI mice. As further revealed by LV pressure-volume relationship analyses, CKO MI mice but not CTL MI mice displayed significant decreases in stroke volume, cardiac output, and the maximum rates of LV pressure rising or declining and of LV volume declining, as well as significant increases in LV end-diastolic pressure and Tau, compared with their respective Sham controls. LC3-II flux assays reveal that autophagic flux is decreased in CKO mouse myocardium as well as in cultured Uchl1-deficient cardiomyocytes. In conclusion, UCHL1 of cardiomyocytes is dispensable for development but promotes macroautophagy in cardiomyocytes. Upregulation of UCHL1 in post-MI hearts occurs primarily in the cardiomyocytes and protects against post-MI cardiac remodeling and malfunction likely through supporting autophagic flux and proteostasis during a stress condition.

A new undesirable genetic factor, neuropathy with splayed forelimbs (JNS), has been identified recently in the Jersey breed. Calves affected with JNS are unable to stand on splayed forelimbs that exhibit significant extensor rigidity and excessive lateral abduction at birth. Affected calves generally are alert at birth but exhibit neurologic symptoms, including spasticity of head and neck and convulsive behavior. Other symptoms reported include dislocated shoulders, congenital craniofacial anomalies, and degenerative myelopathy. Inheritance of an undesirable genetic factor was determined from a study of 16 affected calves reported by Jersey breeders across the United States. All of their pedigrees traced back on both paternal and maternal sides to a common ancestor born in 1995. Genotypes revealed that JNS is attributable to a specific haplotype on Bos taurus autosome 6. Currently 8.2% of the genotyped US Jersey population are carriers of the haplotype. Sequencing of the region of shared homozygosity revealed missense variant rs1116058914 at base 60,158,901 of the ARS-UCD1.2 reference map as the most concordant with the genetic condition and the most likely cause. The single-base G to A substitution is in the coding region of the last exon of UCHL1 , which is conserved across species. Mutations in humans and gene knockouts in mice cause similar recessive symptoms and muscular degeneration. Since December 2020, carrier status has been tracked using the identified haplotype and reported for all 459,784 genotyped Jersey animals. With random mating, about 2,200 affected calves per year with losses of about $250,000 would result from the 1.3 million US Jersey cows in the national population. Selection and mating programs can reduce numbers of JNS-affected births using either the haplotype status or a direct gene test in the future. Breeders should report calf abnormalities to their breed association to help discover new defects such as JNS. [ABSTRACT FROM AUTHOR]

Abstract Background Parkinson’s disease (PD) is the second most common neurodegenerative disorder. PD associated human UCHL1 (Ubiquitin C-terminal hydrolase L1) gene belongs to the family of deubiquitinases and is known to be highly expressed in neurons (1–2% in soluble form). Several functions of UCHL1 have been proposed including ubiquitin hydrolyze activity, ubiquitin ligase activity and stabilization of the mono-ubiquitin. Mutations in human UCHL1 gene have been associated with PD and other neurodegenerative disorders. The present study aims to decipher the sequence evolutionary pattern and structural dynamics of UCHL1. Furthermore, structural and interactional analysis of UCHL1 was performed to help elucidate the pathogenesis of PD. Results The phylogenetic tree topology suggests that the UCHL1 gene had originated in early gnathostome evolutionary history. Evolutionary rate analysis of orthologous sequences reveals strong purifying selection on UCHL1. Comparative structural analysis of UCHL1 pinpoints an important protein segment spanning amino acid residues 32 to 39 within secretion site with crucial implications in evolution and PD pathogenesis through a well known phenomenon called intragenic epistasis. Identified critical protein segment appears to play an indispensable role in protein stability, proper protein conformation as well as harboring critical interaction sites. Conclusions Conclusively, the critical protein segment of UCHL1 identified in the present study not only demonstrates the relevant role of intraprotein conformational epistasis in the pathophysiology of PD but also offers a novel therapeutic target for the disease.

Abstract Background Despite advancements over the past decade revealing molecular aberrations characteristic of endometrial cancer (EC) subtypes, serous ECs remain difficult to treat and associated with poor outcomes. This is due, in part, to the rarity of these tumors within clinical trials and the inability to directly target the most frequent genomic abnormalities. One of the most commonly somatically mutated genes in serous ECs is the tumor suppressor F‐box and WD repeat domain containing 7 (FBXW7). Methods To identify changes in protein expression associated with FBXW7 mutation, we clustered regularly interspaced short palindromic repeats (CRISPR)‐edited ARK4 FBXW7 nonmutant serous EC cells to insert recurrent FBXW7 mutations. We then compared the liquid chromatography tandem mass spectrometry‐based proteomic profiles of CRISPR‐edited ARK1 and ARK4 serous EC cells to matched parental cells. Results Among distinct total and phosphorylated proteins that were significantly differentially expressed in FBXW7‐mutant cell lines compared to matched parental lines, we identified increased PADI2 (peptidyl arginine deiminase 2) expression in all ARK1 and ARK4 CRISPR‐edited FBXW7‐mutant cell lines. We further confirmed the correlation between FBXW7 mutation and increased PADI2 expression in a third biological background, JHUEM‐1 endometrioid EC cells. Finally, we established that PADI2 protein is expressed in primary serous endometrial tumors. Conclusion Our findings provide novel insight into proteomic changes associated with FBXW7 mutation in serous ECs and identify PADI2 as a novel potential therapeutic target for these tumors.

Uterine leiomyomas are smooth-muscle tumors originating in the myometrium and are the most common pelvic tumors in women of reproductive age. Symptomatic tumors may result in abnormal uterine bleeding, bladder dysfunction, pelvic discomfort, and reproductive issues, such as infertility and miscarriage. There are currently few non-invasive treatments for leiomyoma, but there are no practical early intervention or preventive methods. In this study, human uterine leiomyoma and myometrial tissues were used to detect the protein and mRNA expression levels of UCHL1. To explore the effects of UCHL1 knockdown and inhibition in leiomyoma and myometrial cells, we determined the mRNA expressions of COL1A1 and COL3A1. Collagen gel contraction and wound-healing assays were performed on myometrial and leiomyoma cells. We found that UCHL1 expression was considerably higher in uterine leiomyomas than in the myometrium. COL1A1 and COL3A1 expression levels were downregulated after inhibition of UCHL1 in human leiomyoma cells. Furthermore, the elimination of UCHL1 significantly decreased the migration and contractility of leiomyoma cells. In conclusion, these results indicate that UCHL1 is involved in the growth of leiomyoma in humans. For the treatment of uterine leiomyoma, targeting UCHL1 activity may be a unique and possible therapeutic strategy.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver disease. At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic. HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation; however, the cellular targets of its deubiquitinating activity are poorly defined. Here, we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis. HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies., (© 2024 The Author(s).)

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. [ABSTRACT FROM AUTHOR]