Ajit Ray

DJ-1 Loss by Glutaredoxin but Not Glutathione Depletion Triggers Daxx Translocation and Cell Death

Environmental and genetic causes are implicated in the etiopathogenesis of Parkinsons disease (PD), a neurodegenerative movement disorder. DJ-1, a putative gene recessively linked to early onset PD, functions as an antioxidant, transcriptional co-activator, and molecular chaperone. We examined DJ-1 status following global perturbation of protein thiol homeostasis by depleting cellular antioxidant glutathione or downregulating glutaredoxin 1, a thiol disulfide oxidoreductase, wherein both paradigms generate oxidative stress. While these perturbations did not affect expression of DJ-1 mRNA, downregulation of glutaredoxin 1 but not glutathione depletion caused loss of DJ-1 protein, translocation of Daxx (a death-associated protein) from nucleus, and cell death. Overexpression of wild-type DJ-1, but not the cysteine mutants, prevented Daxx translocation and cytotoxicity. Protease inhibitors prevented constitutive DJ-1 loss. Residual DJ-1 was present in reduced state, indicating that DJ-1 when oxidized was degraded through proteolysis. Thus, loss of DJ-1 occurring through its oxidative modification and subsequent proteolysis mediated through dysregulation of thiol disulfide oxidoreductase may contribute to pathogenesis of sporadic PD, thus providing a link between environmental challenges and constitutive levels of this vital protein.

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells

The hypoxic bone marrow (BM) microenvironment confers growth/survival and drug resistance in multiple myeloma (MM) cells. Novel therapies targeting the MM cell in its hypoxic BM milieu may overcome drug resistance. Recent studies led to the development of a novel molecule RRx-001 with hypoxia-selective epigenetic and nitric oxide-donating properties. Here, we demonstrate that RRx-001 decreases the viability of MM cell lines and primary patient cells, as well as overcomes drug resistance. RRx-001 inhibits MM cell growth in the presence of BM stromal cells. RRx-001-induced apoptosis is associated with: (i) activation of caspases; (ii) release of ROS and nitrogen species; (iii) induction of DNA damage via ATM/γ-H2AX; and (iv) decrease in DNA methyltransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. The deubiquitylating enzyme USP7 stimulates DNMT1 activity, and conversely, USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. RRx-001 plus USP7 inhibitor P5091 triggered synergistic anti-MM activity. MM xenograft studies show that RRx-001 is well tolerated, inhibits tumor growth and enhances survival. Combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Our results provide the rationale for translation of RRx-001, either alone or in combination, to clinical evaluation in MM.

Regulation of DJ-1 by Glutaredoxin 1 in Vivo: Implications for Parkinson’s Disease

Parkinsons disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx(-/-) mice we provide show that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homologue in a C. elegans in vivo model of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.

MPTP activates ASK1-p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model.

Activation of apoptosis signal-regulating kinase 1 (ASK1)-p38 MAPK death signaling cascade is implicated in the death of dopaminergic neurons in substantia nigra in Parkinsons disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream p38 MAPK in a time-dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thioredoxin (Trx1), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase µ 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein covariation network analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mediated TNF signaling through intrathecal administration of TNF-neutralizing antibody prevented Trx1 oxidation and downstream ASK1-p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1-p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states.

A novel agent SL-401 induces anti-myeloma activity by targeting plasmacytoid dendritic cells, osteoclastogenesis and cancer stem-like cells

Novel therapies for multiple myeloma (MM) can target mechanism(s) in the host-MM bone marrow (BM) microenvironment mediating MM progression and chemoresistance. Our studies showed increased numbers of tumor-promoting, immunosuppressive and drug-resistant plasmacytoid dendritic cells (pDCs) in the MM BM microenvironment. pDC-MM cell interactions upregulate interleukin-3 (IL-3), which stimulates both pDC survival and MM cell growth. Since IL-3 R is highly expressed on pDCs in the MM BM milieu, we here targeted pDCs using a novel IL-3 R-targeted therapeutic SL-401. In both in vitro and in vivo models of MM in its BM milieu, SL-401 decreases viability of pDCs, blocks pDC-induced MM cell growth, and synergistically enhances anti-MM activity of bortezomib and pomalidomide. Besides promoting pDC survival and MM cell growth, IL-3 also mediates progression of osteolytic bone disease in MM. Osteoclast (OCL) progenitor cells express IL-3 R, and we show that SL-401 abrogates monocyte-derived OCL formation and bone resorption. Finally, we show that SL-401 also decreases the viability of IL-3 R-expressing cancer stem-like cells in MM. Overall, our study provides the preclinical basis for clinical trials of SL-401 to block pDC-induced MM cell growth, inhibit osteoclastogenesis and target MM stem-like cell subpopulations to improve patient outcome in MM.

Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease

Dendritic spine loss is recognized as an early feature of Alzheimers disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression. SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimers disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

Estimation of the critical viscous sublayer in heat transfer problems

Imagining a disturbance made on a compressible boundary layer with the help of a heat source, the critical viscous sublayer, through which the skin friction at any point on a surface is connected with the heat transferred from a heated element embedded in it, has been estimated. Under similar conditions of external flow (Ray1)) the ratio of the critical viscous sublayer to the undisturbed boundary layer thickness is about one-tenth in the laminar case and one hundredth in the turbulent case. These results are similar to those (cf.1)) found in shock wave boundary layer interaction problems.

CHAPTER 8. Redox Signalling in Dopaminergic Cell Death and Survival

Oxidative stress has been demonstrated to be a key pathogenic mechanism for the relatively selective degeneration of dopaminergic neurons in substantia nigra pars compacta seen in Parkinsons disease. But, it remains to be fully resolved how pathological features as widespread and generalized as oxidative stress can lead to cell-specific changes. This can potentially occur if specific cell-signalling pathways are triggered in response to oxidative stress only in certain cells, culminating in neuronal death. In this chapter, we discuss how dopaminergic neurons are more susceptible to oxidative stress and reactive oxygen species generation due to their cellular and molecular phenotype, and by putative mechanisms of redox-regulated repression of the Akt cell survival signalling pathway and activation of the ASK1 death signalling pathway. We present evidence from cell culture and animal models and in autopsy samples from Parkinsons disease patients. Future studies are needed to determine how these pathways interact with other Parkinsons disease associated pathological mechanisms to give us a more comprehensive understanding of the disease process.

LOSS OF F-ACTIN IN SYNAPTOSOMES CORRELATES WITH COGNITIVE DYSFUNCTION (BRAAK STAGING, B-AMYLOID LOAD AND TANGLE LOAD) IN PATIENTS WITH MCI AND AD

protein that is known to define the dendritic spine morphology. It is also involved in synaptic plasticity that mediates contextual fear conditioning. The primary objective of this study was to delineate the molecular mechanisms underlying spine loss and behavioral dysfunction seen early in AD. Methods:We used APP/PS1DE9 mice (APP/PS1) and litter mate WT controls for our experiments. Contextual fear conditioning was used to assess associative fear learning and memory. We used an innovative method to isolate highly enriched F-Actin and G-Actin fractions from synaptosomes from cortices of 1, 2, 4 month old (pre-plaque phase) and 9 month old APP/PS1 mice and age matched controls. We also utilized AMS-derivatization approach to determine the reduced form of F-actin. Statistical comparisons were performed using unpaired t-tests or ANOVA followed by posthoc test. Results: We found significant decrease in F-actin levels in the synaptosomes from 1 month old APP/PS1 mice and this reduction was sustained until 9 months when overt symptoms are observed. While, F-actin levels were unaffected in post-nuclear supernatant across the ages examined. We observed loss of reduced F-actin but not reduced G-actin in the synaptosomes of APP/PS1 mice. Synaptosomal actin-Sglutathionylation, which is known to hinder F-actin polymerization, was significantly increased in 1 month old APP/PS1 mice. Interestingly the contextual fear conditioning behavioral deficits seen in APP/PS1 mice was reversed by a single intrathecal dose (500 ng/25 gram mouse) of Jasplakinolide, an actin polymerizing agent. Further, latrunculin A (500 ng/25 gram mouse), which promotes actin depolymerization induced contextual fear conditioning deficits in WT mice. Conclusions: We demonstrate that the cytoskeletal organization of F-actin is perturbed in synaptic compartment early in AD and leads to behavioral deficits, which can be reversed by actin polymerizing agents. Our results indicate that F-actin is an early target in the pathogenesis of AD.