Identification of a conserved protein motif in a group of growth factor receptors

AbstractResidues 370–383 (helix C) of the human nerve growth factor receptor (NGF-R) are highly similar to the sequence of the 14 residue wasp toxin, mastoparan. Both regions are predicted to form amphiphilic α-helices, as is the amino-terminal region of the third intracytoplasmic loop (i3) of the β2-adrenergic receptor (β2AR). As both mastoparan and the β2AR i3 interact with G-proteins, it is suggested that helix C of the NGF-R may facilitate interactions with a cytoplasmic protein. A similar structural motif was identified in the cytoplasmic domains of a number of other growth factor receptors, suggesting an important role for this motif in signal transduction mechanisms

Modulation of inducible nitric oxide synthase expression by sumoylation

<p>Abstract</p> <p>Background</p> <p>In astrocytes, the inflammatory induction of Nitric Oxide Synthase type 2 (NOS2) is inhibited by noradrenaline (NA) at the transcriptional level however its effects on specific transcription factors are not fully known. Recent studies show that the activity of several transcription factors including C/EBPβ, which is needed for maximal NOS2 expression, is modulated by conjugation of the small molecular weight protein SUMO. We examined whether the expression of SUMO Related Genes (SRGs: SUMO-1, the conjugating enzyme Ubc9, and the protease SENP1) are affected by inflammatory conditions or NA and whether SUMO-1 regulates NOS2 through interaction with C/EBPβ.</p> <p>Methods</p> <p>Bacterial endotoxin lipopolysaccharide (LPS) was used to induce inflammatory responses including NOS2 expression in primary astrocytes. The mRNA levels of SRGs were determined by QPCR. A functional role for SUMOylation was evaluated by determining effects of over-expressing SRGs on NOS2 promoter and NFκB binding-element reporter constructs. Interactions of SUMO-1 and C/EBPβ with the NOS2 promoter were examined by chromatin immunoprecipitation assays. Interactions of SUMO-1 with C/EBPβ were examined by immunoprecipitation and Western blot analysis and by fluorescence resonance energy transfer (FRET) assays.</p> <p>Results</p> <p>LPS decreased mRNA levels of SUMO-1, Ubc9 and SENP1 in primary astrocytes and a similar decrease occurred during normal aging in brain. NA attenuated the LPS-induced reductions and increased SUMO-1 above basal levels. Over-expression of SUMO-1, Ubc9, or SENP1 reduced the activation of a NOS2 promoter, whereas activation of a 4 × NFκB binding-element reporter was only reduced by SUMO-1. ChIP studies revealed interactions of SUMO-1 and C/EBPβ with C/EBP binding sites on the NOS2 promoter that were modulated by LPS and NA. SUMO-1 co-precipitated with C/EBPβ and a close proximity was confirmed by FRET analysis.</p> <p>Conclusion</p> <p>Our results demonstrate that SUMOylation regulates NOS2 expression in astrocytes, and point to modification of C/EBPβ as a possible mechanism of action. Targeting the SUMOylation pathway may therefore offer a novel means to regulate inflammatory NOS2 expression in neurological conditions and diseases.</p

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

Abstract Background Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes. Methods Primary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFα, IFNγ, and IL-1β) or by LPS plus IFNγ. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included. Results In this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies.</p

Neuronal Conditional Knockout of Collapsin Response Mediator Protein 2 Ameliorates Disease Severity in a Mouse Model of Multiple Sclerosis

We previously showed that treatment with lanthionine ketimine ethyl ester (LKE) reduced disease severity and axonal damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis and increased neuronal maturation and survival in vitro. A major target of LKE is collapsin response mediator protein 2 (CRMP2), suggesting this protein may mediate LKE actions. We now show that conditional knockout of CRMP2 from neurons using a CamK2a promoter to drive Cre recombinase expression reduces disease severity in the myelin oligodendrocyte glycoprotein (MOG)35\u201355 EAE model, associated with decreased spinal cord axonal damage, and less glial activation in the cerebellum, but not the spinal cord. Immunohistochemical staining and quantitative polymerase chain reaction show CRMP2 depletion from descending motor neurons in the motor cortex, but not from spinal cord neurons, suggesting that the benefits of CRMP2 depletion on EAE may stem from effects on upper motor neurons. In addition, mice in which CRMP2 S522 phosphorylation was prevented by substitution for an alanine residue also showed reduced EAE severity. These results show that modification of CRMP2 expression and phosphorylation can influence the course of EAE and suggests that treatment with CRMP2 modulators such as LKE act in part by reducing CRMP2 S522 phosphorylation

Phospho-mTOR expression in human glioblastoma microglia-macrophage cells.

Abstract The glioblastoma (GBM) immune microenvironment is highly heterogeneous, and microglia may represent 30–70% of the entire tumor. However, the role of microglia and other specific immune populations is poorly characterized. Activation of mTOR signaling occurs in numerous human cancers and has roles in microglia-glioma cell interactions. We now show in human tumor specimens (42 patients), that 39% of tumor-associated microglial (TAM) cells express mTOR phosphorylated at Ser-2448; and similar mTOR activation is observed using a human microglia-glioma interaction paradigm. In addition, we confirm previous studies that microglia express urea and ARG1 (taken as M2 marker) in the presence of glioma cells, and this phenotype is down-regulated in the presence of a mTOR inhibitor. These results suggest that mTOR suppression in GBM patients might induce a reduction of the M2 phenotype expression in up to 40% of all TAMs. Since the M2 profile of microglial activation is believed to be associated with tumor progression, reductions in that phenotype may represent an additional anti-tumor mechanism of action of mTOR inhibitors, along with direct anti-proliferative activities

PPAR-γ Thiazolidinedione Agonists and Immunotherapy in the Treatment of Brain Tumors

Thiazolidinediones (TZDs) are selective agonists of the peroxisome proliferator-activated receptor (PPAR) gamma, a transcription factor belonging to the superfamily of nuclear hormone receptors. Although activation of PPARγ by TZDs has been best characterized by its ability to regulate expression of genes associated with lipid metabolism, PPARγ agonists have other physiological effects including modulating pro- and anti-inflammatory gene expression and inducing apoptosis in several cell types including glioma cells and cell lines. Immunotherapeutic approaches to reducing brain tumors are focused on means to reduce the immunosuppressive responses of tumors which dampen the ability of cytotoxic T-lymphocytes to kill tumors. Initial studies from our lab show that combination of an immunotherapeutic strategy with TZD treatment provides synergistic benefit in animals with implanted tumors. The potential of this combined approach for treatment of brain tumors is reviewed in this report

Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1β production

BACKGROUND: Under pathological conditions, microglia produce proinflammatory mediators which contribute to neurologic damage, and whose levels can be modulated by endogenous factors including neurotransmitters such as norepinephrine (NE). We investigated the ability of NE to suppress microglial activation, in particular its effects on induction and activity of the inducible form of nitric oxide synthase (NOS2) and the possible role that IL-1β plays in that response. METHODS: Rat cortical microglia were stimulated with bacterial lipopolysaccharide (LPS) to induce NOS2 expression (assessed by nitrite and nitrate accumulation, NO production, and NOS2 mRNA levels) and IL-1β release (assessed by ELISA). Effects of NE were examined by co-incubating cells with different concentrations of NE, adrenergic receptor agonists and antagonists, cAMP analogs, and protein kinase (PK) A and adenylate cyclase (AC) inhibitors. Effects on the NFκB:IκB pathway were examined by using selective a NFκB inhibitor and measuring IκBα protein levels by western blots. A role for IL-1β in NOS2 induction was tested by examining effects of caspase-1 inhibitors and using caspase-1 deficient cells. RESULTS: LPS caused a time-dependent increase in NOS2 mRNA levels and NO production; which was blocked by a selective NFκB inhibitor. NE dose-dependently reduced NOS2 expression and NO generation, via activation of β2-adrenergic receptors (β2-ARs), and reduced loss of inhibitory IkBα protein. NE effects were replicated by dibutyryl-cyclic AMP. However, co-incubation with either PKA or AC inhibitors did not reverse suppressive effects of NE, but instead reduced nitrite production. A role for IL-1β was suggested since NE potently blocked microglial IL-1β production. However, incubation with a caspase-1 inhibitor, which reduced IL-1β levels, had no effect on NO production; incubation with IL-receptor antagonist had biphasic effects on nitrite production; and NE inhibited nitrite production in caspase-1 deficient microglia. CONCLUSIONS: NE reduces microglial NOS2 expression and IL-1β production, however IL-1β does not play a critical role in NOS2 induction nor in mediating NE suppressive effects. Changes in magnitude or kinetics of cAMP may modulate NOS2 induction as well as suppression by NE. These results suggest that dysregulation of the central cathecolaminergic system may contribute to detrimental inflammatory responses and brain damage in neurological disease or trauma

The legacy of redlining in the effect of foreclosures on Detroit residents’ self-rated health

Historical practices, such as housing discrimination in Detroit, have been shown to have lasting impacts on communities. Perhaps the most explicit example is the practice of redlining in the 1930s, whereby lenders outlined financially undesirable neighborhoods, populated by minority families, on maps and prevented residents from moving to better resourced neighborhoods. Awareness of historical housing discrimination may improve research assessing the impacts of current neighborhood characteristics on health. Using the Detroit Neighborhood Health Study (DNHS), we assessed the association between two-year changes in home foreclosure rates following the 2007–2008 Great Recession, and residents’ five-year self-rated health trajectories (2008–2013); and estimated the confounding bias introduced by ignoring historical redlining practices in the city. We used both ecological and multilevel models to make inference about person- and community-level processes. In a neighborhood-level linear regression adjusted for confounders (including percent redlined); a 10%-point slower foreclosure rate recovery was associated with an increase in prevalence of poor self-rated health of 0.31 (95% CI:−0.02 to 0.64). At the individual level, it was associated with a within-person increase in probability of poor health of 0.45 (95% CI:0.15–0.72). Removing redlining from the model biased the estimated effect upward to 0.38 (95% CI:0.07–0.69) and 0.56 (95% CI:0.21–0.84) in the neighborhood and individual-level models, respectively. Stratum-specific foreclosure recovery effects indicate stronger influence in neighborhoods with a greater proportion of residents identifying as white and a greater degree of historic redlining. These findings support earlier theory suggesting a historical influence of structural discrimination on the association between current neighborhood characteristics and health, and suggests that historical redlining specifically may increase vulnerability to contemporary neighborhood foreclosures. Community interventions should consider historical discrimination in conjunction with current place-based indicators to more equitably improve population health

Epinephrine Impairs Lipid Resuscitation from Bupivacaine Overdose

Background Lipid emulsion infusion reverses local anesthetic-induced cardiac toxicity, but the effect of adding epinephrine has not been studied. We compared escalating doses of epinephrine on recovery with lipid infusion in a rat model of bupivacaine overdose. Methods Rats anesthetized with isoflurane received an IV bolus of 20 mg/kg bupivacaine, producing asystole (zero time) in all animals. Ventilation (100% oxygen) and chest compressions were started immediately, and at 3 min the rats received one of six IV treatments (n = 5 for all groups): 5 ml/kg saline followed by infusion for 2 min at 1.0 ml x kg x min, and a second 5 ml/kg bolus at 5 min; or the same bolus and infusion treatment using 30% lipid emulsion plus a single injection of epinephrine at one of five doses: 0 (lipid control), 1, 2.5, 10, or 25 mcg/kg. An electrocardiogram and arterial pressure were monitored continuously, and arterial blood gas was measured at 7.5 and 15 min. Results Epinephrine improved initial return of spontaneous circulation (rate-pressure product &gt; 30% baseline) but only 3 of 5 rats at 10 mcg/kg and 1 of 5 rats at 25 mcg/kg sustained return of spontaneous circulation by 15 min. Lipid alone resulted in slower but more sustained recovery. Epinephrine doses above a threshold near 10 mcg/kg increased lactate, worsened acidosis, and resulted in poor recovery at 15 min, as compared with lipid controls. There was tight correlation of epinephrine dose to serum lactate at 15 min. Conclusions Epinephrine over a threshold dose near 10 mcg/kg impairs lipid resuscitation from bupivacaine overdose, possibly by inducing hyperlactatemia

Effect of pioglitazone treatment on behavioral symptoms in autistic children

INTRODUCTION: Autism is complex neuro-developmental disorder which has a symptomatic diagnosis in patients characterized by disorders in language/communication, behavior, and social interactions. The exact causes for autism are largely unknown, but is has been speculated that immune and inflammatory responses, particularly those of Th2 type, may be involved. Thiazolidinediones (TZDs) are agonists of the peroxisome proliferator activated receptor gamma (PPARγ), a nuclear hormone receptor which modulates insulin sensitivity, and have been shown to induce apoptosis in activated T-lymphocytes and exert anti-inflammatory effects in glial cells. The TZD pioglitazone (Actos) is an FDA-approved PPARγ agonist used to treat type 2 diabetes, with a good safety profile, currently being tested in clinical trials of other neurological diseases including AD and MS. We therefore tested the safety and therapeutic potential of oral pioglitazone in a small cohort of children with diagnosed autism. CASE DESCRIPTION: The rationale and risks of taking pioglitazone were explained to the parents, consent was obtained, and treatment was initiated at either 30 or 60 mg per day p.o. A total of 25 children (average age 7.9 ± 0.7 year old) were enrolled. Safety was assessed by measurements of metabolic profiles and blood pressure; effects on behavioral symptoms were assessed by the Aberrant Behavior Checklist (ABC), which measures hyperactivity, inappropriate speech, irritability, lethargy, and stereotypy, done at baseline and after 3–4 months of treatment. DISCUSSION AND EVALUATION: In a small cohort of autistic children, daily treatment with 30 or 60 mg p.o. pioglitazone for 3–4 months induced apparent clinical improvement without adverse events. There were no adverse effects noted and behavioral measurements revealed a significant decrease in 4 out of 5 subcategories (irritability, lethargy, stereotypy, and hyperactivity). Improved behaviors were inversely correlated with patient age, indicating stronger effects on the younger patients. CONCLUSION: Pioglitazone should be considered for further testing of therapeutic potential in autistic patients