A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation

<p>Abstract</p> <p>Background</p> <p>Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells.</p> <p>Results</p> <p>RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated <it>in vitro </it>in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination <it>in vitro</it>. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both <it>in vitro </it>and <it>in vivo</it>, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.</p> <p>Conclusion</p> <p>Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.</p

From the ECM to the Cytoskeleton and Back: How Integrins Orchestrate T Cell Action

T lymphocytes constitute a highly dynamic tissue type. During the course of their lives, they travel through a variety of physiological environments and experience a multitude of interactions with extracellular matrix components and other cells. In order to do this, they must receive many environmental cues, and translate these signals into the appropriate biological actions. Particularly dramatic are the cytoskeletal shape changes a T cell must undergo during the processes of leaving the bloodstream, migrating through tissues, and encountering antigen. In this review, we highlight the role of integrins in providing a link between the extracellular environment and cytoskeletal regulation and how these receptors help to orchestrate T cell migration and antigen recognition

Chronic Intermittent Hypoxia Impairs Baroreflex Control Of Heart Rate But Enhances Heart Rate Responses To Vagal Efferent Stimulation In Anesthetized Mice

Chronic intermittent hypoxia (CIH) leads to increased sympathetic nerve activity and arterial hypertension. In this study, we tested the hypothesis that CIH impairs baroreflex (BR) control of heart rate (HR) in mice, and that decreased cardiac chronotropic responsiveness to vagal efferent activity contributes to such impairment. C57BL/6J mice were exposed to either room air (RA) or CIH (6-min alternations of 21% O2 and 5.7% O2, 12 h/day) for 90 days. After the treatment period, mice were anesthetized (Avertin) and arterial blood pressure (ABP) was measured from the femoral artery. Mean ABP (MABP) was significantly increased in mice exposed to CIH (98.7 ± 2.5 vs. RA: 78.9 ± 1.4 mmHg, P \u3c 0.001). CIH increased HR significantly (584.7 ± 8.9 beats/min; RA: 518.2 ± 17.9 beats/min, P \u3c 0.05). Sustained infusion of phenylephrine (PE) at different doses (0.1-0.4 μg/min) significantly increased MABP in both CIH and RA mice, but the ABP-mediated decreases in HR were significantly attenuated in mice exposed to CIH (P \u3c 0.001). In contrast, decreases in HR in response to electrical stimulation of the left vagus nerve (30 μA, 2-ms pulses) were significantly enhanced in mice exposed to CIH compared with RA mice at low frequencies. We conclude that CIH elicits a sustained impairment of baroreflex control of HR in mice. The blunted BR-mediated bradycardia occurs despite enhanced cardiac chronotropic responsiveness to vagal efferent stimulation. This suggests that an afferent and/or a central defect is responsible for the baroreflex impairment following CIH

A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation-4

Lected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. . Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. . Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). . Percentage of cell death before and after transfection with transfection reagent removed 6 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). . Percentage of cell death of the control and 24 h transfected samples subjected to a 7 h OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). . Assessment of the siRNA silencing efficiency. RNA samples were collected 24 and 48 h after transfection with siRNAs. Down regulation of RNF182 mRNA was analyzed by RT-PCR. In: lanes 1 and 3 – 24 and 48 h after transfection with RNF182 siRNAs, lanes 2 and 4 – 24 and 48 h after transfection with non-targeting pool negative control siRNAs, respectively. . Percentage of cell death 24 h after siRNA transfection, with or with out OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.005; t-test). C24 – 24 h after transfection with non-targeting pool negative control siRNAs, siRNA24 – 24 h after transfection with mouse RNF182 on-target plus smart pool siRNAs, c24-OGD – 24 h after transfection with non-targeting pool negative control siRNAs plus OGD treatment, siRNA24-OGD – 24 h after transfection with on-target plus smart pool siRNAs plus OGD treatment.<p><b>Copyright information:</b></p><p>Taken from "A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation"</p><p>http://www.molecularneurodegeneration.com/content/3/1/4</p><p>Molecular Neurodegeneration 2008;3():4-4.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2279130.</p><p></p

A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation-0

He open reading frame (black bar) and 3' untranslated region of both transcripts are solely encoded by exon 4. Structural motifs of the encoded protein were predicted by ExPASy tools.<p><b>Copyright information:</b></p><p>Taken from "A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation"</p><p>http://www.molecularneurodegeneration.com/content/3/1/4</p><p>Molecular Neurodegeneration 2008;3():4-4.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2279130.</p><p></p

A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation-5

Anti-ubiquitin antibody. The GST-SIAH-1 protein was used as a positive control.<p><b>Copyright information:</b></p><p>Taken from "A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation"</p><p>http://www.molecularneurodegeneration.com/content/3/1/4</p><p>Molecular Neurodegeneration 2008;3():4-4.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2279130.</p><p></p

A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation-3

D mRNA of 4 AD and 5 age-matched control subjects. The value of the control sample was set at 100%. The percentage of the AD sample was calculated by 100x 2, where ΔCt is the cycle number difference between the AD sample and the control sample. The experiments were performed in triplicate. Asterisk indicates a significant difference (ρ < 0.005; t-test). . Changes in mRNA levels of RNF182 transcript in individual control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from mRNA of 5 AD and 5 age-matched control subjects. The qRT-PCR results were calculated against the average result (control mean) of the control samples, set at 100%. Percentage of each sample was calculated by 100x 2, where ΔCt is the cycle number difference between each sample and the control mean. The experiments were performed in triplicate. . Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD and OGD with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). . Changes in RNF182 protein levels in NT2 neurons treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-RNF182 antibody using 120 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. . Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD plus 20 μM β-amyloid peptide and OGD plus 20 μM β-amyloid peptide with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). Insert: Nuclear morphology of OGD plus Aβ treated cells was examined under an Olympus B x 50 fluorescence microscope after fixing and staining the cells with DAPI.<p><b>Copyright information:</b></p><p>Taken from "A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation"</p><p>http://www.molecularneurodegeneration.com/content/3/1/4</p><p>Molecular Neurodegeneration 2008;3():4-4.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2279130.</p><p></p