The depletion of ubiquilin in Drosophila melanogaster disturbs neurochemical regulation to drive activity and behavioral deficits.

Drosophila melanogaster is a useful and highly tractable model organism for understanding the molecular mechanisms of human diseases. We previously characterized a new dUbqn knockdown model that induces learning-memory and locomotive deficits mediated by impaired proteostasis. Although proteinopathies are the main causes of neurodegenerative diseases, limited information is currently available on the relationship between proteostasis and neurodegenerative-related behavioral perturbations, such as locomotion, wakefulness, and sexual activities. Thus, the present study aimed to elucidate the mechanisms by which dUbqn depletion which is known to cause proteinopathies, affects neurodegenerative-related behavioral perturbations. Pan-neuronal dUbqn-depleted flies showed significantly reduced evening activity along with altered pre- and postsynaptic structural NMJ's proteins by attenuating signals of Bruchpilot puncta and GluRIIA clustering. In addition, the neurochemical profiles of GABA, glutamate, dopamine, and serotonin were disturbed and these changes also affected courtship behaviors in dUbqn-depleted flies. Collectively, these results extend our understanding on how dUbqn depletion affects neurochemical regulation to drive behavioral disturbances that are generally found in the early stage of neurodegenerative diseases. Moreover, the present study may contribute a novel finding to the design of new agents that prevent disease progression or even treat diseases related to neurodegeneration.

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.

Disruption of polyubiquitin gene Ubb causes dysregulation of neural stem cell differentiation with premature gliogenesis.

Disruption of polyubiquitin gene Ubb leads to early-onset reactive gliosis and adult-onset hypothalamic neurodegeneration in mice. However, it remains unknown why reduced levels of ubiquitin (Ub) due to loss of Ubb lead to these neural phenotypes. To determine whether or not the defects in neurons or their progenitors per se, but not in their cellular microenvironment, are the cause of the neural phenotypes observed in Ubb(-/-) mice, we investigated the properties of cultured cells isolated from Ubb(-/-) mouse embryonic brains. Although cells were cultured under conditions promoting neuronal growth, Ubb(-/-) cells underwent apoptosis during culture in vitro, with increased numbers of glial cells and decreased numbers of neurons. Intriguingly, at the beginning of the Ubb(-/-) cell culture, the number of neural stem cells (NSCs) significantly decreased due to their reduced proliferation and their premature differentiation into glial cells. Furthermore, upregulation of Notch target genes due to increased steady-state levels of Notch intracellular domain (NICD) led to the dramatic reduction of proneuronal gene expression in Ubb(-/-) cells, resulting in inhibition of neurogenesis and promotion of gliogenesis. Therefore, our study suggests an unprecedented role for cellular Ub pools in determining the fate and self-renewal of NSCs.

Cellular ubiquitin pool dynamics and homeostasis.

Ubiquitin (Ub) is a versatile signaling molecule that plays important roles in a variety of cellular processes. Cellular Ub pools, which are composed of free Ub and Ub conjugates, are in dynamic equilibrium inside cells. In particular, increasing evidence suggests that Ub homeostasis, or the maintenance of free Ub above certain threshold levels, is important for cellular function and survival under normal or stress conditions. Accurate determination of various Ub species, including levels of free Ub and specific Ub chain linkages, have become possible in biological specimens as a result of the introduction of the proteomic approach using mass spectrometry. This technology has facilitated research on dynamic properties of cellular Ub pools and has provided tools for in-depth investigation of Ub homeostasis. In this review, we have also discussed the consequences of the disruption of Ub pool dynamics and homeostasis via deletion of polyubiquitin genes or mutations of deubiquitinating enzymes. The common consequence was a reduced availability of free Ub and a significant impact on the function and viability of cells. These observations further indicate that the levels of free Ub are important determinants for cellular protection.

Ubiquitin B in cervical cancer: critical for the maintenance of cancer stem-like cell characters.

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.

Mutant ubiquitin (UBB+1) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3).

Mutant ubiquitin (UBB(+1)) accumulates in the hallmarks of tauopathies and polyglutamine diseases. We show that the deubiquitinating enzyme YUH1 of Saccharomyces cerevisiae and its mouse and human ortholog UCH-L3 are able to hydrolyze the C-terminal extension of UBB(+1). This yields another dysfunctional ubiquitin molecule (UB(G76Y)) with biochemical properties similar to full length UBB(+1). UBB(+1) may be detected in post-mortem tissue due to impaired C-terminal truncation of UBB(+1). Although the level of UCH-L3 protein in several neurodegenerative diseases is unchanged, we show that in vitro oxidation of recombinant UCH-L3 impairs its deubiquitinating activity. We postulate that impaired UCH-L3 function may contribute to the accumulation of full length UBB(+1) in various pathologies.

Altered testicular gene expression patterns in mice lacking the polyubiquitin gene Ubb.

Ubiquitin (Ub) is an essential protein found in all eukaryotic cells and plays important roles in a variety of cellular functions including germ cell development. We have previously reported that targeted disruption of the polyubiquitin gene Ubb results in male and female infertility in Ubb(-/-) mice, with germ cells arrested at meiotic prophase I. Although reduced Ub levels in germ cells are believed to be responsible for the fertility defect in Ubb(-/-) mice, it is still unclear how reduced Ub levels result in sterility. Here we describe the results of a microarray analysis of the murine testicular transcriptome, which demonstrates dramatically altered gene expression patterns in Ubb(-/-) mice, possibly related to reduced levels of histone 2A (H2A) ubiquitylation. We find that large numbers of genes related to fertility, metabolism, transcription, and the ubiquitin-proteasome system (UPS) are misregulated in Ubb(-/-) mice. Such wide-ranging alterations in gene expression suggest that loss of the Ubb gene does not mimic a single-gene defect phenotype, but instead may affect gene expression more globally. These dramatic changes in gene expression could, at least in part, contribute to the complex fertility and metabolic phenotypes seen in these mice.

Loss of polyubiquitin gene Ubb leads to metabolic and sleep abnormalities in mice.

AIMS: Ubiquitin performs essential roles in a myriad of signalling pathways required for cellular function and survival. Recently, we reported that disruption of the stress-inducible ubiquitin-encoding gene Ubb reduces ubiquitin content in the hypothalamus and leads to adult-onset obesity coupled with a loss of arcuate nucleus neurones and disrupted energy homeostasis in mice. Neuropeptides expressed in the hypothalamus control both metabolic and sleep behaviours. In order to demonstrate that the loss of Ubb results in broad hypothalamic abnormalities, we attempted to determine whether metabolic and sleep behaviours were altered in Ubb knockout mice. METHODS: Metabolic rate and energy expenditure were measured in a metabolic chamber, and sleep stage was monitored via electroencephalographic/electromyographic recording. The presence of neurodegeneration and increased reactive gliosis in the hypothalamus were also evaluated. RESULTS: We found that Ubb disruption leads to early-onset reduced activity and metabolic rate. Additionally, we have demonstrated that sleep behaviour is altered and sleep homeostasis is disrupted in Ubb knockout mice. These early metabolic and sleep abnormalities are accompanied by persistent reactive gliosis and the loss of arcuate nucleus neurones, but are independent of neurodegeneration in the lateral hypothalamus. CONCLUSIONS: Ubb knockout mice exhibit phenotypes consistent with hypothalamic dysfunction. Our data also indicate that Ubb is essential for the maintenance of the ubiquitin levels required for proper regulation of metabolic and sleep behaviours in mice.

A genomewide RNAi screen for genes that affect the stability, distribution and function of P granules in Caenorhabditis elegans.

P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their "germ granule" counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells.

Hypothalamic neurodegeneration and adult-onset obesity in mice lacking the Ubb polyubiquitin gene.

Nearly all neurodegenerative diseases are associated with abnormal accumulation of ubiquitin (Ub) conjugates within neuronal inclusion bodies. To directly test the hypothesis that depletion of cellular Ub is sufficient to cause neurodegeneration, we have disrupted Ubb, one of four genes that supply Ub in the mouse. Here, we report that loss of Ubb led to a progressive degenerative disorder affecting neurons within the arcuate nucleus of the hypothalamus. This neurodegenerative cytopathology was accompanied by impaired hypothalamic control of energy balance and adult-onset obesity. Ubb was highly expressed in vulnerable hypothalamic neurons and total Ub levels were selectively reduced in the hypothalamus of Ubb-null mice. These findings demonstrate that maintenance of adequate supplies of cellular Ub is essential for neuronal survival and establish that decreased Ub availability is sufficient to cause neuronal dysfunction and death.

Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination.

Dendritic cells have a unique function in the immune response owing to their ability to stimulate immunologically naive T lymphocytes. In response to microbial and inflammatory stimuli, dendritic cells enhance their capacity for antigen presentation by a process of terminal differentiation, termed maturation. The conversion of immature to mature dendritic cells is accompanied by a marked cellular reorganization, including the redistribution of major histocompatibility complex class II molecules (MHC II) from late endosomal and lysosomal compartments to the plasma membrane and the downregulation of some forms of endocytosis, which has been thought to slow the clearance of MHC II from the surface. The relative extent to which these or other mechanisms contribute to the regulation of surface MHC II remains unclear, however. Here we find that the MHC II beta-chain cytoplasmic tail is ubiquitinated in mouse immature dendritic cells. Although only partly required for the sequestration of MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II ceased upon maturation, resulting in the accumulation of MHC II at the cell surface. Dendritic cells thus exhibit a unique ability to regulate MHC II surface expression by selectively controlling MHC II ubiquitination.

Neuropeptide research discloses part of the secrets of Alzheimer's disease neuropathogenesis: state of the art 2004.

Molecular misreading, a process discovered in the late 1990s, entails the formation of aberrant transcripts due to the inaccurate conversion of genomic information, and results in an accumulation of aberrant proteins. The aberrant transcripts are formed as a result of a dinucleotide deletion (e.g. DeltaGA, DeltaGU) during or after transcription. Either the RNA polymerase starts to make mistakes (e.g. stuttering) in simple sequence repeats, such as GAGAG, or erroneous editing of transcripts occurs. If these aberrant transcripts are not detected and degraded efficiently, they can be translated from the deletion onwards into the +1 reading frame. The resulting proteins are therefore called +1 proteins. If functional domains are located downstream of the frameshift site, the result will be a protein with a potential loss or gain of function. It has been hypothesized that quality control mechanisms for both transcripts and proteins work less efficiently during aging, which is why +1 proteins may become manifest and contribute to age-related diseases in neuronal and non-neuronal cells.