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1.  Activation of the Ubiquitin Proteasome Pathway in a Mouse Model of Inflammatory Myopathy 
Arthritis and rheumatism  2013;65(12):3248-3258.
Myositis is characterized by severe muscle weakness. We and others have previously shown that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of myositis. The present study was undertaken to identify perturbed pathways and assess their contribution to muscle disease in a mouse myositis model.
Stable isotope labeling with amino acids in cell culture (SILAC) was used to identify alterations in the skeletal muscle proteome of myositic mice in vivo. Differentially altered protein levels identified in the initial comparisons were validated using a liquid chromatography tandem mass spectrometry spike-in strategy and further confirmed by immunoblotting. In addition, we evaluated the effect of a proteasome inhibitor, bortezomib, on the disease phenotype, using well-standardized functional, histologic, and biochemical assessments.
With the SILAC technique we identified significant alterations in levels of proteins belonging to the ER stress response, ubiquitin proteasome pathway (UPP), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. We validated the myositis-related changes in the UPP and demonstrated a significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1) in myositis, but not in muscle affected by other dystrophies or normal muscle. Inhibition of the UPP with bortezomib significantly improved muscle function and also significantly reduced tumor necrosis factor α expression in the skeletal muscle of mice with myositis.
Our findings indicate that ER stress activates downstream UPPs and contributes to muscle degeneration and that UCHL-1 is a potential biomarker for disease progression. UPP inhibition offers a potential therapeutic strategy for myositis.
PMCID: PMC4080828  PMID: 24022788
3.  The molecular basis of skeletal muscle weakness in a mouse model of inflammatory myopathy 
Arthritis and rheumatism  2012;64(11):3750-3759.
It is generally believed that muscle weakness in patients with polymyositis and dermatomyositis is due to autoimmune and inflammatory processes. However, it has been observed that there is a poor correlation between the suppression of inflammation and a recovery of muscle function in patients. We have therefore hypothesized that non-immune mechanisms also contribute to muscle weakness. In particular, it has been suggested that an acquired deficiency of AMP deaminase (AMPD1) may be responsible for muscle weakness in myositis.
We have used comprehensive functional, behavioral, histological, molecular, enzymatic and metabolic assessments before and after the onset of inflammation in MHC class I mouse model of autoimmune inflammatory myositis.
We found that muscle weakness and metabolic disturbances were detectable in the mice prior to the appearance of infiltrating mononuclear cells. Force contraction analysis of muscle function revealed that weakness was correlated with AMDP1 expression and was myositis-specific. We also demonstrated that decreasing AMPD1 expression results in decreased muscle strength in healthy mice. Fiber typing suggested that fast-twitch muscles are converted to slow-twitch muscles as myositis progresses, and microarray results indicated that AMPD1 and other purine nucleotide pathway genes are suppressed, along with genes essential to glycolysis.
These data suggest that an AMPD1 deficiency is acquired prior to overt muscle inflammation and is responsible, at least in part, for the muscle weakness that occurs in the mouse model of myositis. AMPD1 is therefore a potential therapeutic target in myositis.
PMCID: PMC3485437  PMID: 22806328
4.  Daily Supplementation of D-ribose Shows No Therapeutic Benefits in the MHC-I Transgenic Mouse Model of Inflammatory Myositis 
PLoS ONE  2013;8(6):e65970.
Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures.
Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose.
Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.
PMCID: PMC3681851  PMID: 23785461
5.  Idiopathic inflammatory myopathies: pathogenic mechanisms of muscle weakness 
Skeletal Muscle  2013;3:13.
Idiopathic inflammatory myopathies (IIMs) are a heterogenous group of complex muscle diseases of unknown etiology. These diseases are characterized by progressive muscle weakness and damage, together with involvement of other organ systems. It is generally believed that the autoimmune response (autoreactive lymphocytes and autoantibodies) to skeletal muscle-derived antigens is responsible for the muscle fiber damage and muscle weakness in this group of disorders. Therefore, most of the current therapeutic strategies are directed at either suppressing or modifying immune cell activity. Recent studies have indicated that the underlying mechanisms that mediate muscle damage and dysfunction are multiple and complex. Emerging evidence indicates that not only autoimmune responses but also innate immune and non-immune metabolic pathways contribute to disease pathogenesis. However, the relative contributions of each of these mechanisms to disease pathogenesis are currently unknown. Here we discuss some of these complex pathways, their inter-relationships and their relation to muscle damage in myositis. Understanding the relative contributions of each of these pathways to disease pathogenesis would help us to identify suitable drug targets to alleviate muscle damage and also improve muscle weakness and quality of life for patients suffering from these debilitating muscle diseases.
PMCID: PMC3681571  PMID: 23758833
Adaptive immune; Autophagy; Cytokines; Endoplasmic reticulum stress; Innate immune; Myositis; Skeletal muscle; TLRs
6.  Endoplasmic Reticulum Stress in Skeletal Muscle Homeostasis and Disease 
Current rheumatology reports  2012;14(3):238-243.
Our appreciation of the role of endoplasmic reticulum(ER) stress pathways in both skeletal muscle homeostasis and the progression of muscle diseases is gaining momentum. This review provides insight into ER stress mechanisms during physiologic and pathological disturbances in skeletal muscle. The role of ER stress in the response to dietary alterations and acute stressors, including its role in autoimmune and genetic muscle disorders, has been described. Recent studies identifying ER stress markers in diseased skeletal muscle are noted. The emerging evidence for ER–mitochondrial interplay in skeletal muscle and its importance during chronic ER stress in activation of both inflammatory and cell death pathways (autophagy, necrosis, and apoptosis) have been discussed. Thus, understanding the ER stress–related molecular pathways underlying physiologic and pathological phenotypes in healthy and diseased skeletal muscle should lead to novel therapeutic targets for muscle disease.
PMCID: PMC3587844  PMID: 22410828
Skeletal muscle; Endoplasmic reticulum; Sarcoplasmic reticulum; Mitochondria; Myositis; ER stress; Autophagy; Necrosis; Apoptosis; Muscle disease
7.  Role of non-immune mechanisms of muscle damage in idiopathic inflammatory myopathies 
Idiopathic inflammatory myopathies (IIMs) comprise a group of autoimmune diseases that are characterized by symmetrical skeletal muscle weakness and muscle inflammation with no known cause. Like other autoimmune diseases, IIMs are treated with either glucocorticoids or immunosuppressive drugs. However, many patients with an IIM are frequently resistant to immunosuppressive treatments, and there is compelling evidence to indicate that not only adaptive immune but also several non-immune mechanisms play a role in the pathogenesis of these disorders. Here, we focus on some of the evidence related to pathologic mechanisms, such as the innate immune response, endoplasmic reticulum stress, non-immune consequences of MHC class I overexpression, metabolic disturbances, and hypoxia. These mechanisms may explain how IIM-related pathologic processes can continue even in the face of immunosuppressive therapies. These data indicate that therapeutic strategies in IIMs should be directed at both immune and non-immune mechanisms of muscle damage.
PMCID: PMC3446443  PMID: 22546362
8.  Localization and Sub-Cellular Shuttling of HTLV-1 Tax with the miRNA Machinery 
PLoS ONE  2012;7(7):e40662.
The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs, suggests that the cellular RNAi machinery is a major means by which the host mounts a defense response against present day retroviruses. Indeed, cellular miRNAs target and hybridize to specific sequences of both HTLV-1 and HIV-1 viral transcripts. However, much like the variety of host immune responses to retroviral infection, the virus itself contains mechanisms that assist in the evasion of viral inhibition through control of the cellular RNAi pathway. Retroviruses can hijack both the enzymatic and catalytic components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active viral infection or promote a latent state. Here, we show that HTLV-1 Tax contributes to the dysregulation of the RNAi pathway by altering the expression of key components of this pathway. A survey of uninfected and HTLV-1 infected cells revealed that Drosha protein is present at lower levels in all HTLV-1 infected cell lines and in infected primary cells, while other components such as DGCR8 were not dramatically altered. We show colocalization of Tax and Drosha in the nucleus in vitro as well as coimmunoprecipitation in the presence of proteasome inhibitors, indicating that Tax interacts with Drosha and may target it to specific areas of the cell, namely, the proteasome. In the presence of Tax we observed a prevention of primary miRNA cleavage by Drosha. Finally, the changes in cellular miRNA expression in HTLV-1 infected cells can be mimicked by the add back of Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus.
PMCID: PMC3393700  PMID: 22808228
9.  Mechanisms of regulation of oligodendrocyte development by p38 MAP kinase 
The Journal of Neuroscience  2010;30(33):11011-11027.
Many extracellular and intrinsic factors regulate oligodendrocyte development, but their signaling pathways remain poorly understood. While the p38 mitogen activated protein kinase (MAPK)-dependent pathway is implicated in oligodendrocyte progenitor cell (OPC) lineage progression, its molecular targets involved in myelinogenesis are largely unidentified. We have analyzed mechanisms by which p38MAPK regulates oligodendrocyte development and demonstrate that p38MAPK inhibition prevents OPC lineage progression, and inhibits MBP promoter activity and Sox10 function. In white matter tissue, differential levels of MAPK phosphorylation are observed in oligodendrocyte lineage cells. Phosphorylated p38MAPK was found in CC1- and CNP-expressing differentiated oligodendrocytes of the adult brain, and was temporally associated with a decline in the levels of phosphorylated ERK in cells of this lineage. PDGF stimulates the phosphorylation of ERK, p38MAPK and JNK, and p38MAPK inhibition was associated with increased ERK, JNK and c-Jun phosphorylation. In the presence of PDGF, simultaneous inhibition of p38MAPK and either MEK or JNK significantly alleviates the repression of myelin gene expression and lineage progression induced by p38MAPK inhibition alone. Dominant negative c-Jun reverses the inhibition of myelin promoter activity by active MEK1 or dominant negative p38MAPKα mutants, and phosphorylated c-Jun was detected at the MBP promoter following p38MAPK inhibition, indicating c-Jun as a negative mediator of p38MAPK action. Our findings indicate that p38MAPK activity in the brain supports myelin gene expression through distinct mechanisms via positive and negative regulatory targets. We show that oligodendrocyte differentiation involves p38-mediated Sox10 regulation and crosstalk with parallel ERK and JNK pathways to repress c-Jun activity.
PMCID: PMC3327889  PMID: 20720108
MAP kinases; myelin; oligodendrocyte; development; cross-talk; gene expression
10.  Novel HIV-1 Therapeutics through Targeting Altered Host Cell Pathways 
Expert opinion on biological therapy  2009;9(11):1369-1382.
The emergence of drug-resistant human immunodeficiency virus type I (HIV-1) strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse-transcriptase, viral protease, or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we will discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host’s proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be performed to better design therapeutics against HIV-1.
PMCID: PMC2785118  PMID: 19732026
11.  microRNA machinery is an integral component of drug-induced transcription inhibition in HIV-1 infection 
RNA interference plays a significant role in manipulating cellular and viral mechanisms to maintain latency during HIV-1 infection. HIV-1 produces several microRNAs including one from the TAR element which alter the host's response to infection. Since cyclin/cdk complexes are important for viral transcription, these studies focus on the possible cdk inhibitors that inhibit viral transcription, without affecting normal cellular mechanisms. Roscovitine and Flavopiridol are well-studied cdk inhibitors that are effective at suppressing their target cdks at a low IC50. These cdk inhibitors and possibly future generations of drugs are affected by microRNA mechanisms. From our studies, we developed a third generation derivative called CR8#13. In cells that lack Dicer there was a higher level of basal viral LTR-reporter transcription. When drugs, specifically Flavopiridol and CR8#13 were added, the transcriptional inhibition of the LTR was less potent in cells that lacked Dicer. Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes). We next asked whether the addition of CR8 or CR8#13 could possibly increase levels of TAR microRNA in HIV-1 LTR containing cells. We demonstrate that the 3'TAR microRNA is produced in higher amounts after drug treatment, resulting in microRNA recruitment to the LTR. MicroRNA recruitment results in chromatin alteration, changes in Pol II phosphorylation and viral transcription inhibition. In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter. With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.
PMCID: PMC2902143  PMID: 20628499
microRNA; HIV-1; TAR; cdk inhibitor; ATP analogs; Tat transactivation
13.  9-aminoacridine Inhibition of HIV-1 Tat Dependent Transcription 
Virology Journal  2009;6:114.
As part of a continued search for more efficient anti-HIV-1 drugs, we are focusing on the possibility that small molecules could efficiently inhibit HIV-1 replication through the restoration of p53 and p21WAF1 functions, which are inactivated by HIV-1 infection. Here we describe the molecular mechanism of 9-aminoacridine (9AA) mediated HIV-1 inhibition. 9AA treatment resulted in inhibition of HIV LTR transcription in a specific manner that was highly dependent on the presence and location of the amino moiety. Importantly, virus replication was found to be inhibited in HIV-1 infected cell lines by 9AA in a dose-dependent manner without inhibiting cellular proliferation or inducing cell death. 9AA inhibited viral replication in both p53 wildtype and p53 mutant cells, indicating that there is another p53 independent factor that was critical for HIV inhibition. p21WAF1 is an ideal candidate as p21WAF1 levels were increased in both p53 wildtype and p53 mutant cells, and p21WAF1 was found to be phosphorylated at S146, an event previously shown to increase its stability. Furthermore, we observed p21WAF1 in complex with cyclin T1 and cdk9 in vitro, suggesting a direct role of p21WAF1 in HIV transcription inhibition. Finally, 9AA treatment resulted in loss of cdk9 from the viral promoter, providing one possible mechanism of transcriptional inhibition. Thus, 9AA treatment was highly efficient at reactivating the p53 – p21WAF1 pathway and consequently inhibiting HIV replication and transcription.
PMCID: PMC2723079  PMID: 19630958
British Medical Journal  1927;1(3453):491.
PMCID: PMC2454063

Results 1-17 (17)