Mild Oxidative Stress Induced by Sodium Arsenite Reduces Lipocalin-2 Expression Levels in Cortical Glial Cells.

Astrocytes and microglia, the most abundant glial cells in the central nervous system, are involved in maintaining homeostasis in the brain microenvironment and in the progression of various neurological disorders. Lipocalin-2 (LCN2) is a small secretory protein that can be transcriptionally upregulated via nuclear factor kappa B (NF-κB) signaling. It is synthesized and secreted by glial cells, resulting in either the restoration of damaged neural tissues or the induction of neuronal apoptosis in a context-dependent manner. It has recently been reported that when glial cells are under lipopolysaccharide-induced inflammatory stress, either reduced production or accelerated degradation of LCN2 can alleviate neurotoxicity. However, the regulatory mechanisms of LCN2 in glial cells are not yet fully understood. In this study, we used primary astroglial-enriched cells which produce LCN2 and found that the production of LCN2 could be reduced by sodium arsenite treatment. Surprisingly, the reduced LCN2 production was not due to the suppression of NF-κB signaling. Mild oxidative stress induced by sodium arsenite treatment activated antioxidant responses and downregulated Lcn2 expression without reducing the viability of astroglial-enriched cells. Intriguingly, reduced LCN2 production could not be achieved by simple activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway in astroglial-enriched cells. Thus, it appears that mild oxidative stress, occurring in an Nrf2-independent manner, is required for the downregulation of Lcn2 expression. Taken together, our findings provide new insights into the regulatory mechanisms of LCN2 and suggest that mild oxidative stress may alter LCN2 homeostasis, even under neuroinflammatory conditions.

Lipocalin-2: a therapeutic target to overcome neurodegenerative diseases by regulating reactive astrogliosis.

Glial cell activation precedes neuronal cell death during brain aging and the progression of neurodegenerative diseases. Under neuroinflammatory stress conditions, lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin or 24p3, is produced and secreted by activated microglia and reactive astrocytes. Lcn2 expression levels are known to be increased in various cells, including reactive astrocytes, through the activation of the NF-κB signaling pathway. In the central nervous system, as LCN2 exerts neurotoxicity when secreted from reactive astrocytes, many researchers have attempted to identify various strategies to inhibit LCN2 production, secretion, and function to minimize neuroinflammation and neuronal cell death. These strategies include regulation at the transcriptional, posttranscriptional, and posttranslational levels, as well as blocking its functions using neutralizing antibodies or antagonists of its receptor. The suppression of NF-κB signaling is a strategy to inhibit LCN2 production, but it may also affect other cellular activities, raising questions about its effectiveness and feasibility. Recently, LCN2 was found to be a target of the autophagy‒lysosome pathway. Therefore, autophagy activation may be a promising therapeutic strategy to reduce the levels of secreted LCN2 and overcome neurodegenerative diseases. In this review, we focused on research progress on astrocyte-derived LCN2 in the central nervous system.

Alleviation of neurotoxicity induced by polystyrene nanoplastics by increased exocytosis from neurons.

Nanoplastics (NPs) are potentially toxic and pose a health risk as they can induce an inflammatory response and oxidative stress at cellular and organismal levels. Humans can be exposed to NPs through various routes, including ingestion, inhalation, and skin contact. Notably, uptake into the body via inhalation could result in brain accumulation, which may occur directly across the blood-brain barrier or via other routes. NPs that accumulate in the brain may be endocytosed into neurons, inducing neurotoxicity. Recently, we demonstrated that exposure to polystyrene (PS)-NPs reduces the viability of neurons. We have also reported that inhibiting the retrograde transport of PS-NPs by histone deacetylase 6 (HDAC6) prevents their intracellular accumulation and promotes their export in mouse embryonic fibroblasts. However, whether HDAC6 inhibition can improve neuronal viability by increasing exocytosis of PS-NPs from neurons remains unknown. In this study, mice were intranasally administered fluorescent PS-NPs (PS-YG), which accumulated in the brain and showed potential neurotoxic effects. In cultured neurons, the HDAC6 inhibitor ACY-1215 reduced the fluorescence signal detected from PS-YG, suggesting that the removal of PS-YG from neurons was promoted. Therefore, these results suggest that blocking the retrograde transport of PS-NPs using an HDAC6 inhibitor can alleviate the neurotoxic effects of PS-NPs that enter the brain.

Reduced secretion of LCN2 (lipocalin 2) from reactive astrocytes through autophagic and proteasomal regulation alleviates inflammatory stress and neuronal damage.

LCN2/neutrophil gelatinase-associated lipocalin/24p3 (lipocalin 2) is a secretory protein that acts as a mammalian bacteriostatic molecule. Under neuroinflammatory stress conditions, LCN2 is produced and secreted by activated microglia and reactive astrocytes, resulting in neuronal apoptosis. However, it remains largely unknown whether inflammatory stress and neuronal loss can be minimized by modulating LCN2 production and secretion. Here, we first demonstrated that LCN2 was secreted from reactive astrocytes, which were stimulated by treatment with lipopolysaccharide (LPS) as an inflammatory stressor. Notably, we found two effective conditions that led to the reduction of induced LCN2 levels in reactive astrocytes: proteasome inhibition and macroautophagic/autophagic flux activation. Mechanistically, proteasome inhibition suppresses NFKB/NF-κB activation through NFKBIA/IκBα stabilization in primary astrocytes, even under inflammatory stress conditions, resulting in the downregulation of Lcn2 expression. In contrast, autophagic flux activation via MTOR inhibition reduced the intracellular levels of LCN2 through its pre-secretory degradation. In addition, we demonstrated that the N-terminal signal peptide of LCN2 is critical for its secretion and degradation, suggesting that these two pathways may be mechanistically coupled. Finally, we observed that LPS-induced and secreted LCN2 levels were reduced in the astrocyte-cultured medium under the above-mentioned conditions, resulting in increased neuronal viability, even under inflammatory stress.Abbreviations: ACM, astrocyte-conditioned medium; ALP, autophagy-lysosome pathway; BAF, bafilomycin A1; BTZ, bortezomib; CHX, cycloheximide; CNS, central nervous system; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; JAK, Janus kinase; KD, knockdown; LCN2, lipocalin 2; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin kinase; NFKB/NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; NFKBIA/IκBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; OVEX, overexpression; SLC22A17, solute carrier family 22 member 17; SP, signal peptide; SQSTM1, sequestosome 1; STAT3, signal transducer and activator of transcription 3; TNF/TNF-α, tumor necrosis factor; TUBA, tubulin, alpha; TUBB3/ß3-TUB, tubulin, beta 3 class III; UB, ubiquitin; UPS, ubiquitin-proteasome system.

Increased clearance of non-biodegradable polystyrene nanoplastics by exocytosis through inhibition of retrograde intracellular transport.

Nanoplastics (NPs) are derived from microplastics and may cause health problems. We previously showed that 100 nm polystyrene (PS)-NPs enter cells, including mouse embryonic fibroblasts (MEFs), and their intracellular accumulation induces inflammatory and oxidative stress. Moreover, PS-NP uptake was found to occur via endocytosis, and they accumulated mostly at the juxtanuclear position, but never within the nucleus. We speculated that PS-NPs were cleared from cells when they were no longer exposed to PS-NPs. However, the effects of PS-NPs on the cellular machinery remain unknown. The accumulation of PS-NPs at the juxtanuclear position may be due to retrograde transport along microtubules. To confirm this, we treated PS-NP-exposed MEFs with inhibitors of histone deacetylase 6 (HDAC6), dynein, or microtubule polymerization and found greatly diminished intracellular and juxtanuclear accumulation. Moreover, rapid clearance of PS-NPs was observed when MEFs were treated with an HDAC6 inhibitor. PS-NPs were removed by exocytosis, as confirmed by treatment with an exocytosis inhibitor. Furthermore, inhibiting the retrograde transport of PS-NPs alleviated the activation of the antioxidant response pathway, inflammatory and oxidative stress, and reactive oxygen species generation. In summary, inhibition of the retrograde transport of non-biodegradable PS-NPs leads to their rapid export by exocytosis, which may reduce their cytotoxicity.

Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development.

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein-protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.

Stress Response of Mouse Embryonic Fibroblasts Exposed to Polystyrene Nanoplastics.

Polystyrene (PS) nanoplastic exposure has been shown to affect the viability of neuronal cells isolated from mouse embryonic brains. However, the viability of mouse embryonic fibroblasts (MEFs) was not affected although PS nanoplastics accumulated in the cytoplasm. It is currently unknown whether MEFs do not respond to PS nanoplastics or their cellular functions are altered without compromising viability. Here, we found that PS nanoplastics entered the cells via endocytosis and were then released into the cytoplasm, probably by endosomal escape, or otherwise remained in the endosome. Oxidative and inflammatory stress caused by intracellular PS nanoplastics induced the antioxidant response pathway and activated the autophagic pathway. However, colocalization of the autophagic marker LC3B and PS nanoplastics suggested that PS nanoplastics in the cytoplasm might interfere with normal autophagic function. Furthermore, autophagic flux could be impaired, probably due to accumulation of PS nanoplastic-containing lysosomes or autolysosomes. Intriguingly, the level of accumulated PS nanoplastics decreased during prolonged culture when MEFs were no longer exposed to PS nanoplastics. These results indicate that accumulated PS nanoplastics are removed or exported out of the cells. Therefore, PS nanoplastics in the cytoplasm affect cellular functions, but it is temporal and MEFs can overcome the stress caused by PS nanoplastic exposure.

Regulation of polyubiquitin genes to meet cellular ubiquitin requirement.

Ubiquitin (Ub) is one of the proteins that are highly conserved from yeast to humans. It is an essential core unit of the welldefined post-translational modification, called ubiquitination, which is involved in a variety of biological processes. In metazoans, Ub is encoded by two monoubiquitin genes and two polyubiquitin genes, in which a single Ub is fused to a ribosomal protein or Ub coding units are arranged in tandem repeats. In mice, polyubiquitin genes (Ubb and Ubc) play a pivotal role to meet the requirement of cellular Ub pools during embryonic development. In addition, expression levels of polyubiquitin genes are increased to adapt to environmental stimuli such as oxidative, heat-shock, and proteotoxic stress. Several researchers have reported about the perturbation of Ub pools through genetic alteration or exogenous Ub delivery using diverse model systems. To study Ub pool changes in a physiologically relevant manner, changing Ub pools via the regulation of endogenous polyubiquitin gene expression has recently been introduced. Furthermore, to understand the regulation of polyubiquitin gene expression more precisely, cis-acting elements and trans-acting factors, which are regulatory components of polyubiquitin genes, have been analyzed. In this review, we discuss how the role of polyubiquitin genes has been studied during the past decade, especially focusing on their regulation. [BMB Reports 2021; 54(4): 189-195].

Neurotoxic potential of polystyrene nanoplastics in primary cells originating from mouse brain.

Polystyrene (PS) and chemically modified compounds in the PS family have long been used in commercial and industrial fields. However, it is poorly understood whether nanoscale-PS microplastic or PS nanoplastic exposure leads to perturbations in fundamental cellular functions, such as proliferation, differentiation, and apoptosis. Herein, we cultured three types of primary cells, including mouse embryonic fibroblasts (MEFs), mixed neuronal cells isolated from embryonic cortex, and cortical astrocytes, and investigated the effects of their exposure to PS nanoplastics with a 100 nm diameter. Although PS nanoplastic exposure did not affect the viability of MEFs or astrocytes, it significantly reduced the viability of mixed neuronal cells. Consistent with the observed effect on cellular viability, levels of the apoptosis marker, cleaved caspase-3, were elevated exclusively in mixed neuronal cells. To investigate whether cells uptake PS nanoplastics into the cytoplasm, we exposed MEFs and neurons to fluorescent PS latex beads and monitored fluorescence over time. We found that PS nanoplastics were deposited and accumulated in the cytoplasm in a concentration-dependent manner. Although astrocytes were not apoptotic upon exposure to PS nanoplastics, they underwent reactive astrocytosis, with increased levels of lipocalin-2 and proinflammatory cytokines. Therefore, our findings suggested that the vulnerability of cells to the deposition and accumulation of PS nanoplastics in the cytoplasm was dependent on cell type. Furthermore, based on our data from primary cells originating from mouse brains, we suggest that reactive astrocytosis may contribute to the neuronal apoptosis seen in defective neurons with PS nanoplastics accumulated in the cell body.

Simultaneous Disruption of Both Polyubiquitin Genes Affects Proteasome Function and Decreases Cellular Proliferation.

The ubiquitin (Ub) proteasome system is important for maintaining protein homeostasis and has various roles in cell signaling, proliferation, and cell cycle regulation. In mammals, Ub is encoded by two monoubiquitin and two polyubiquitin genes. Although reduced levels of Ub due to the disruption of one polyubiquitin gene are known to decrease cell proliferation, the effect of disrupting both polyubiquitin genes remains elusive. Polyubiquitin gene Ubc knockout mice are embryonically lethal and polyubiquitin gene Ubb knockout mice are infertile. Thus, it is difficult to study the effects of double knockouts (DKOs). In the present study, the CRISPR/Cas9 system was used to simultaneously knockout both polyubiquitin genes, UBB and UBC, in HEK293T and HeLa cells. In DKO cells, growth decreased significantly compared to the control cells. We observed reduced proteasome function and reduced levels of free Ub in DKO cells. However, the levels of purified proteasome were not different between control and DKO cells, although the mRNA levels of proteasomal subunits were significantly increased in latter. We propose that the reduction of Ub levels, by disruption of both polyubiquitin genes, resulted in an altered proteasomal status, leading to the reduced proteasome activity, and decreased cellular proliferation.

Reduced free ubiquitin levels and proteasome activity in cultured neurons and brain tissues treated with amyloid beta aggregates.

Neurodegenerative diseases are characterized by progressive cognitive decline and the loss of neurons in the central nervous system; many are also characterized by abnormal aggregation of misfolded proteins. Ubiquitin (Ub) is a eukaryotic protein that plays pivotal roles in protein degradation and cellular signaling. Ubiquitinated aggregates are observed in neurodegenerative diseases; this ultimately results in reduced levels of available or free Ub. However, it remains unclear whether neurotoxicity arises from the aggregates or a deficiency of free Ub. To investigate this, we treated primary neurons of mouse embryonic brains with amyloid beta (Aß) 42 and found that free Ub levels were decreased and cell viability was reduced in Aß42-treated neurons. As reduced levels of free Ub are closely related to impaired function of the proteasome, we evaluated proteasome activity and found that proteasome activity was reduced upon treatment of primary neurons and mouse brain slices with Aß42. Therefore, we conclude that proteotoxic stress from Aß42 treatment reduced the levels of available Ub and decreased proteasome activity, resulting in inflammatory stress and apoptosis of neurons.

Disruption of the polyubiquitin gene Ubb reduces the self-renewal capacity of neural stem cells.

Ubiquitin (Ub) is a highly conserved eukaryotic protein that plays pivotal roles in cellular signal transduction, differentiation, and proteolysis. Although we have previously reported that disruption of the polyubiquitin gene Ubb is associated with the dysregulated differentiation of neural stem cells (NSCs) into neurons, it is unclear how gene expression patterns are altered in Ubb knockout (KO) NSCs, and whether this altered gene expression contributes to Ubb KO neural phenotypes. To answer these questions, we used RNA-Seq to compare the transcriptomes of Ubb KO NSCs and Ubb heterozygous (HT) controls. We found that the expression levels of most proliferation markers were decreased in Ubb KO NSCs. To determine whether the reduced levels of proliferation markers were due to reduced self-renewal of NSCs, such as radial glia, we measured the levels of the radial glia marker, Pax6, in mouse embryonic brains at 14.5 dpc. We found that Pax6 levels were decreased and the ventricular zone was thinner in the embryonic brains of Ubb KO mice compared to those of wild-type (WT) control mice. To determine whether the decreased self-renewal of Ubb KO NSCs was caused by cell-autonomous defects and not due to their microenvironment, we transplanted NSCs into WT mouse brains using a cannula system. In mouse brain sections, immunoreactivity of the NSC marker, nestin, was much lower in Ubb KO NSCs than in Ubb HT controls. Therefore, our data suggest that cell-autonomous defects, due to the disruption of Ubb, lead to a decrease in the self-renewal capacity of NSCs and may contribute to their dysregulated differentiation into neurons.

Integrin αvß3-Akt signalling plays a role in radioresistance of melanoma.

Melanoma is a deadly type of skin cancer that is particularly difficult to treat owing to its resistance to radiation therapy. Here, we attempted to determine the key proteins responsible for melanoma radioresistance, with the aim of improving disease response to radiation therapy. Two melanoma cell lines, SK-Mel5 and SK-Mel28, with different radiosensitivities were analysed via RNA-Seq (Quant-Seq) and target proteins with higher abundance in the more radioresistant cell line, SK-Mel28, identified. Among these proteins, integrin αvß3, a well-known molecule in cell adhesion, was selected for analysis. Treatment of SK-Mel28 cells with cilengitide, an integrin αvß3 inhibitor, as well as γ-irradiation resulted in more significant cell death than γ-irradiation alone. In addition, Akt, a downstream signal transducer of integrin αvß3, showed high basic activation in SK-Mel28 and was significantly decreased upon co-treatment with cilengitide and γ-irradiation. MK-2206, an Akt inhibitor, exerted similar effects on the SK-Mel28 cell line following γ-irradiation. Our results collectively demonstrate that the integrin αvß3-Akt signalling pathway contributes to radioresistance in SK-Mel28 cells, which may be manipulated to improve therapeutic options for melanoma.

Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System.

Ubiquitin is required under both normal and stress conditions. Under stress conditions, upregulation of the polyubiquitin gene UBC is essential to meet the requirement of increased ubiquitin levels to confer stress resistance. However, UBC upregulation is usually observed only under stress conditions and not under normal conditions. Therefore, it has not been possible to upregulate UBC under normal conditions to study the effect of excess ubiquitin on cellular machinery. Recently, the CRISPR/Cas9 system has been widely used in biological research as a useful tool to study gene disruption effects. In this study, using an inducible CRISPR/Cas9 variant, a dCas9-VP64 fusion protein, combined with a single guide RNA (sgRNA) containing MS2 aptamer loops and MS2-p65-HSF1, we developed a system to increase the ubiquitin pool via upregulation of UBC. Although it is challenging to upregulate the expression of a gene that is already expressed at high levels, the significance of our system is that UBC upregulation can be induced in an efficient, reversible manner that is compatible with cellular processes, even under normal conditions. This system can be used to study ubiquitin pool dynamics and it will be a useful tool in identifying the role of ubiquitin under normal and stress conditions.

Disruption of the polyubiquitin gene Ubb causes retinal degeneration in mice.

We have previously demonstrated that a reduction in ubiquitin (Ub) levels via disruption of the polyubiquitin gene Ubb results in reactive gliosis and hypothalamic neurodegeneration in mice. However, it is not known whether other neural tissues, apart from the brain, can also be affected by Ubb disruption. We examined the retina, which, being derived from the diencephalon, has the same developmental origin as the hypothalamus. We found that expression levels of Ubb were much higher than those of the other polyubiquitin gene Ubc in the retina. In retinal tissues from Ubb knockout (KO) mice, we found that Ubc expression was upregulated to compensate for the loss of Ubb; however, the Ub pool remained disrupted, with reduced levels of free Ub. To directly demonstrate whether the disrupted Ub pools affect neural integrity in retinal tissues, we investigated retinal layers in control and Ubb KO mice. Using optical coherence tomography and histological analysis, we demonstrated that the thickness of the outer nuclear layer of the retina was decreased in Ubb KO mice compared to control mice, suggesting that retinal degeneration was induced by Ub deficiency. Furthermore, the mRNA and protein levels of rhodopsin decreased and those of glial fibrillary acidic protein increased in Ubb KO mouse retinas. Therefore, the maintenance of Ub pools in the retina appears to be crucial for the survival of photoreceptor cells and the prevention of excessive glial cell activation.

Cytoprotective role of ubiquitin against toxicity induced by polyglutamine-expanded aggregates.

Ubiquitin (Ub) homeostasis is important for cellular function and survival, especially under stress conditions. Recently, we have demonstrated that Ubc-/- (Ub-deficient) mouse embryonic fibroblasts (MEFs) exhibited reduced viability under oxidative stress induced by arsenite, which was not due to dysregulation of the antioxidant response pathway, but rather due to the potential toxicity caused by the misfolded protein aggregates. However, it is still not clear whether Ub deficiency is directly related to the accumulation of toxic protein aggregates, as arsenite itself triggers protein aggregation and renders cells into aberrant conditions such as reduced proteasome function and inhibition of autophagic flux. Therefore, under arsenite treatment, the outcome could be derived from the combination of multiple defective pathways. Furthermore, it has also been suggested that ubiquitination status of misfolded proteins may not be important for the formation of inclusion bodies composed of misfolded protein aggregates. We therefore wondered whether Ub deficiency is sufficient to trigger the accumulation of toxic protein aggregates inside the cells. In this study, we ectopically expressed polyQ-expanded aggregates (Q103) in MEFs and observed inclusion body formation at the juxtanuclear region, which was independent of cellular Ub levels. In contrast to arsenite treatment, polyQ expression did not affect proteasome function. However, we observed an increased accumulation of Q103 aggregates in Ubc-/- MEFs, which was due to impaired autophagic clearance. Finally, we demonstrated that the increased accumulation of Q103 aggregates under Ub deficiency dramatically reduced the viability of cells. Therefore, our results suggest that the maintenance of proper levels of cellular Ub is important to protect cells against the toxicity induced by the accumulation of protein aggregates.

Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro.

Reduced levels of cellular ubiquitin (Ub) pools due to disruption of the polyubiquitin gene Ubb lead to dysregulation of neural stem cell (NSC) differentiation and impaired neuronal maturation in cells isolated from Ubb -/- mouse embryonic brains. However, it is currently unknown whether Ub is required for the specific stage of neuronal development or whether it plays a pleiotropic role throughout the process. To answer this question, we aimed to downregulate Ubb expression temporally during neuronal development, which could not be achieved in Ubb -/- cells. Therefore, we exploited lentivirus-mediated knockdown (KD) of Ubb at different stages of neuronal development, and investigated their phenotypes. Here, we report the outcome of Ubb KD on two independent culture days in vitro (DIV): DIV1 and DIV7. We observed that NSCs did not differentiate properly via Ubb KD on DIV1, but the maturation of already differentiated neurons was intact via Ubb KD on DIV7. Intriguingly, Ubb KD activated Notch signaling when it had been suppressed, but exerted no effect when it had already been activated. Therefore, our study suggests that Ub plays a pivotal role in NSC differentiation to suppress Notch signaling, but not in the subsequent maturation stages of neurons that had already been differentiated.

Effect of p62/SQSTM1 polyubiquitination on its autophagic adaptor function and cellular survival under oxidative stress induced by arsenite.

Oxidative stress induced by arsenite [As(III)] affects protein folding and results in increased levels of misfolded proteins or protein aggregates. Accumulation of misfolded protein aggregates may act as a cue signal for the oligomerization of the autophagic adaptor protein p62, which facilitates recognition of misfolded protein aggregates that are polyubiquitinated with K63 linkages. However, as the autophagic flux is impaired under exposure to As(III), p62 oligomers cannot be cleared by autophagy and accumulate as aggregates with Keap1. This results in the sequestration of Keap1 and the stabilization of Nrf2, which activates the non-canonical Nrf2-Keap1 pathway as an antioxidant response. In this study, we found that polyubiquitination of p62 itself increased upon exposure to As(III) to prevent further oligomerization of p62 and to increase the availability of functional free monomeric p62. We also found that monomeric p62 could also interact with ubiquitinated proteins and that the forced dimerization of p62 was sufficient to increase the interactions with ubiquitinated proteins, probably polyubiquitinated with K63 linkages. Upon exposure to As(III), (1) inability to form oligomeric p62 because of a mutation in the PB1 dimerization domain, or (2) reduced capability to generate monomeric p62 owing to diminished polyubiquitination of p62 itself, resulted in reduced viability of cells. Therefore, upon exposure to As(III), p62 initially needs to form oligomers to activate an antioxidant response pathway. Subsequently, p62 is polyubiquitinated to prevent further oligomerization and ensure the availability of free p62 monomers. We propose that the polyubiquitination of p62 under exposure to As(III) plays an important role in overcoming the impaired autophagic flux by regulating the oligomerization status of p62.

Regulation of REST levels overcomes dysregulation of neural stem cell differentiation caused by disruption of polyubiquitin gene Ubb.

Reduced levels of cellular ubiquitin (Ub) caused by disruption of the polyubiquitin gene Ubb lead to dysregulated differentiation of neural stem/progenitor cells (NSCs) and apoptosis in cells cultured in vitro. However, the underlying mechanisms responsible for these phenotypes in Ub-deficient cells have not been studied extensively. In the present study, we found that levels of repressor element-1 silencing transcription factor (REST) are elevated in Ubb-/- cells. To determine whether dysregulation of NSC differentiation is caused by the increased REST levels, we investigated the effect of reduced REST levels in Ubb-/- cells. Rest knockdown was found to increase the expression of the neuronal marker ßIII-tubulin (TUJ1) and restore the expression pattern of the early neuronal marker α-internexin (α-INX) in Ubb-/- cells. Furthermore, Rest knockdown reduced Ub deficiency-induced apoptosis in cells cultured in vitro. Therefore, our study validates that cellular Ub levels are crucial for precise control of the levels of regulatory proteins such as REST during neurogenesis. We propose that regulation of Rest levels is a promising approach to overcome dysregulation of NSC differentiation caused by disruption of the polyubiquitin gene Ubb.