A Systems Biology Approach towards Deciphering the Unfolded Protein Response in Huntington's Disease

Although the disease causing gene huntingtin has been known for some time, the exact cause of neuronal cell death during _Huntington's disease_ (HD) remains unknown. One potential mechanism contributing to the massive loss of neurons in HD brains might be the _Unfolded Protein Response_ (UPR) which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). As an adaptive response, UPR upregulates transcription of chaperones, temporarily attenuating new translation and activates protein degradation via the proteasome. However, at high levels of ER stress, UPR signalling can contribute to neuronal apoptosis.

Our primary aims include (a) construction of the UPR signalling network, (b) curation and bioinformatical identification of UPR target genes and finally (c) examination of HD gene expression data sets for UPR transcriptional signatures and differential regulation of UPR pathways.

The UPR signalling pathway is reconstructed based on literature review and using the "Unified Interactome database":http://www.unihi.org. Lists of UPR target genes detected by previous experiments or as predicted by computational analysis are compiled. This allows us to perform enrichment analysis for differential HD gene expression and to assess whether UPR expression signatures are prominent during HD pathogenesis.

Results: The canonical UPR pathway is complemented with additional protein interaction data allowing us to assess its embedding into the cellular context and to identify potential modifiers as well as novel drug targets.

Conclusions: The in depth systems biology analysis can give us valuable insights about the involvement of the UPR in HD.
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The Unfolded Protein Response and its potential role in Huntington's disease

Huntington's disease (HD) is a progressive, neurodegenerative disease with fatal outcome. Although the disease-causing gene (huntingtin) has been known for some time, the exact cause of neuronal cell death is still unknown. One potential mechanism contributing to the massive loss of neurons in the brain of HD patients might be the unfolded protein response (UPR), which is activated by accumulation of misfolded proteins in the endoplasmatic reticulum (ER). As an adaptive response to counter-balance accumulation of un- or misfolded proteins, the UPR upregulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. However, it is known that persistent ER stress and activated UPR can cause cell death by triggering of apoptosis. Nevertheless, the evidence linking UPR with HD progression remains inconclusive. Here, we present first analyses of UPR activation during HD based on available expression data. To elucidate the potential role of UPR as a disease-relevant process, we examine its connection to cell death and inflammatory processes. Due to the complexity of these molecular mechanisms, a systems biology approach was pursued

Tubular cell phenotype in HIV-associated nephropathy: Role of phospholipid lysophosphatidic acid

Collapsing glomerulopathy and microcysts are characteristic histological features of HIV-associated nephropathy (HIVAN). We have previously reported the role of epithelial mesenchymal transition (EMT) in the development of glomerular and tubular cell phenotypes in HIVAN. Since persistent tubular cell activation of NF kappa B has been reported in HIVAN, we now hypothesize that HIV may be contributing to tubular cell phenotype via lysophosphatidic acid (LPA) mediated downstream signaling. Interestingly, LPA and its receptors have also been implicated in the tubular interstitial cell fibrosis (TIF) and cyst formation in autosomal dominant polycystic kidney disease (PKD). Primary human proximal tubular cells (HRPTCs) were transduced with either empty vector (EV/HRPTCs), HIV (HIV/HRPTCs) or treated with LPA (LPA/HRPTC). Immunoelectrophoresis of HIV/HRPTCs and LPA/HRPTCs displayed enhanced expression of pro-fibrotic markers: a) fibronectin (2.25 fold), b) connective tissue growth factor (CTGF; 4.8 fold), c) alpha-smooth muscle actin (alpha-SMA; 12 fold), and d) collagen 1(5.7 fold). HIV enhanced tubular cell phosphorylation of ILK-1, FAK, PI3K, Akt, ERKs and P38 MAPK HIV increased tubular cell transcriptional binding activity of NF-kappa B; whereas, a LPA biosynthesis inhibitor (AACOCF3), a DAG kinase inhibitor, a LPA receptor blocker (Ki16425), a NF-kappa B inhibitor (PDTC) and NF kappa B-siRNA not only displayed downregulation of a NF kappa B activity but also showed attenuated expression of profibrotic/EMT genes in HIV milieu. These findings suggest that LPA could be contributing to HIV-induced tubular cell phenotype via NF kappa B activation in HIVAN. (C) 2015 Elsevier Inc. All rights reserved

5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) attenuates the expression of LPS- and Aβ peptide-induced inflammatory mediators in astroglia

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) pathology shows characteristic 'plaques' rich in amyloid beta (Aβ) peptide deposits. Inflammatory process-related proteins such as pro-inflammatory cytokines have been detected in AD brain suggesting that an inflammatory immune reaction also plays a role in the pathogenesis of AD. Glial cells in culture respond to LPS and Aβ stimuli by upregulating the expression of cytokines TNF-α, IL-1β, and IL-6, and also the expression of proinflammatory genes iNOS and COX-2. We have earlier reported that LPS/Aβ stimulation-induced ceramide and ROS generation leads to iNOS expression and nitric oxide production in glial cells. The present study was undertaken to investigate the neuroprotective function of AICAR (a potent activator of AMP-activated protein kinase) in blocking the pro-oxidant/proinflammatory responses induced in primary glial cultures treated with LPS and Aβ peptide.</p> <p>Methods</p> <p>To test the anti-inflammatory/anti-oxidant functions of AICAR, we tested its inhibitory potential in blocking the expression of pro-inflammatory cytokines and iNOS, expression of COX-2, generation of ROS, and associated signaling following treatment of glial cells with LPS and Aβ peptide. We also investigated the neuroprotective effects of AICAR against the effects of cytokines and inflammatory mediators (released by the glia), in blocking neurite outgrowth inhibition, and in nerve growth factor-(NGF) induced neurite extension by PC-12 cells.</p> <p>Results</p> <p>AICAR blocked LPS/Aβ-induced inflammatory processes by blocking the expression of proinflammatory cytokine, iNOS, COX-2 and MnSOD genes, and by inhibition of ROS generation and depletion of glutathione in astroglial cells. AICAR also inhibited down-stream signaling leading to the regulation of transcriptional factors such as NFκB and C/EBP which are critical for the expression of iNOS, COX-2, MnSOD and cytokines (TNF-α/IL-1β and IL-6). AICAR promoted NGF-induced neurite growth and reduced neurite outgrowth inhibition in PC-12 cells treated with astroglial conditioned medium.</p> <p>Conclusion</p> <p>The observed anti-inflammatory/anti-oxidant and neuroprotective functions of AICAR suggest it as a viable candidate for use in treatment of Alzheimer's disease.</p

Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition.

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist-, miR193a inhibitor-, and interferon-γ-induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA-silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship

Rapamycin-induced modulation of HIV gene transcription attenuates progression of HIVAN

HIV-associated nephropathy (HIVAN) is the manifestation of HIV genes expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV genes expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5 mg/kg/day, intraperitoneally) for eight weeks. At the end of experimental period, kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/ cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-kB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects

AT(1)R blockade in adverse milieus: role of SMRT and corepressor complexes

ANG II type 1 receptor blockade (AT(1)R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT(1)R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT(1)R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT(1)R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT(1)R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT(1)R-BLK-induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT(1)R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT(1)R-BLK. We conclude that AT(1)R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)(2)D-deficient states; on the other hand, AT(1)R-BLK contributes to liganded VDR expression in 1,25(OH)(2)D-sufficient states