Hidden Steps Traced in Path to Inflammation

A new mechanism in the inflammation pathway has been identified by HMS researchers working in mice. Specific adenosine receptors inactivated a subunit of the E3-SCF ubiquitin ligase complex, which prevented activation of the pro-inflammatory transcription factor nuclear factor kappa B (NF-kappa B). The findings could advance tissue-specific anti-inflammatory therapies, according to the study, reported in the March issue of the Journal of Clinical Investigation.

Disconnection. HMS researchers discovered that adenosine exerts its anti-inflammatory actions by uncoupling—or deneddylating—CUL-1. In typical inflammatory responses, neddylated CUL-1 and its accompanying E3-SCF complex activate pro-inflammatory NF-kappa B by turning off its inhibitor I kappa B.

Joseph Khoury, a cell and molecular biologist and a former HMS instructor; Juan Ibla, HMS instructor in anesthesia at Children’s Hospital Boston; and colleagues used a model involving oxygen deprivation to induce inflammation in the lungs. In mice, 10 minutes of breathing five percent oxygen induces hypoxia and increases markers of inflammation, including NF-kappa B.

Activation of NF-kappa B can be suppressed by habituating the mice to low oxygen levels with a regimen of hypoxia preconditioning. The mice receive three 10-minute sessions in a chamber containing eight percent oxygen prior to their exposure to the hypoxic five percent oxygen treatment. Preconditioning prolongs survival in the otherwise deadly hypoxic environment. Though the adaptive properties of the regimen make it a good model for studying endogenous protection from inflammation, its molecular mechanisms have been unclear.

Analyzing lung tissue using microarrays and high-performance liquid chromatography, the researchers found that hypoxia preconditioning decreased NF-kappa B expression and promoted the release of the anti-inflammatory molecule adenosine. Based on previous work showing that adenosine directly suppresses NF-kappa B, the team sought to identify other players in the pathway. In particular, they investigated adenosine’s relationship to the uncoupling, or deneddylation, of the Cullin-1 (Cul-1) subunit in the E3-SCF complex, which activates NF-kappa B by turning off its inhibitor I kappa B. The researchers found that adenosine dose-dependently deneddylated Cul-1 from Nedd8, an effect they mimicked with an adenosine receptor analog. Overexpression of individual adenosine receptors revealed that A2B and, to a lesser extent, A1 receptors were particularly involved in the preconditioning-induced suppression of NF-kappa B. Other studies indicate that A2B receptors can protect against bone marrow and vascular inflammation, but not in chronic inflammatory conditions.

To verify their in vitro findings that adenosine causes Cul-1 deneddylation, Khoury, Ibla, and their colleagues compared lung water content, a measure of inflammation, between wild-type mice and mutants lacking the critical enzyme for adenosine formation. Following hypoxia preconditioning, the visibly swollen lungs of the mutant mice had about a 136 percent increase in water content compared to the wild types’ lungs. Immunoblotting revealed that preconditioning offered no protection against hypoxia-induced NF-kappa B expression and higher levels of neddylated Cul-1 in the adenosine-deficient mice.

Deneddylation equals anti-inflammation, summarized Khoury and Ibla of their novel finding that adenosine’s anti-inflammatory actions are due at least in part to deneddylating Cul-1.

—Molly McElroy

Spurring Protein Breakdown in Brain Cells Halts Huntington’s Pathology

HMS researchers have used gene therapy to prolong the survival of striatal cells modeled for Huntington’s disease. The therapy targeted the diseased cells’ impaired ability to degrade mutated huntingtin protein, which can accumulate and become toxic. By overexpressing a particular subunit on proteasomes, the neuroscientists increased protein degradation and made cells more resistant to chemical stressors. The findings are reported in the Feb. 28 issue of PloS ONE.

McLean researcher Ole Isacson, HMS professor of neurology, previously found low proteasome activity in brain and skin cells from Huntington’s patients. “Clearly it seemed the proteolytic activity of the system was reduced all over the brain and in skin,” said Isacson of the 2004 study published in Annals of Neurology. But the researchers did not know why the Huntington’s cells had impaired protein breakdown.

In many ways proteasomes look and behave like garbage disposals. The protein-chopping organelle has a hollow core that traps proteins targeted for recycling. Bladelike enzymes line the core while enzyme gates called proteasome activators (PAs) hasten the movement of the targeted proteins into the catalyzing core. In the 2004 study, Isacson increased protein degradation in healthy skin cells by increasing function of the PA28 enzyme with the overexpression of its alpha and beta subunits. But the PA28 subunit treatment did not work in skin cells from Huntington’s patients. The team wondered whether a different subunit would be effective in Huntington’s disease cells and whether striatal ­neurons—where the huntingtin mutation inflicts the most damage—would be sensitive to the protein-disposing acceleration of PA treatment.

To devise ways to boost protein clearance in Huntington’s, Isacson obtained striatal cells derived from mouse stem cells that were modeled for the disease by collaborator Elena Cattaneo, professor of pharmacy at the University of Milan. Like their 2004 findings in Huntington’s skin cells, the PA28-alpha and -beta subunit treatments did not increase proteasome activity in the Huntington-modeled striatal cells. Yet treatment with a different subunit, PA28-gamma, did work in these brain cells. “The findings show us that the system is specific and that simply increasing any proteolytic activity inside the cell may not work for the disease,” Isacson said.

Next, the proteasome-boosting PA28-gamma treatment was combined with exposure to toxic compounds, including glutamate agonists. By stressing the Huntington’s striatal cells with such compounds, the researchers saw that the PA28-gamma treatment protected against excitoxicity and other pathologically stressing events. The findings indicate that proteasome activation through gene therapy can provide an afflicted cell with a tool to combat its mutation.

Protein aggregates are a common pathological feature in many age-related neurodegenerative diseases, including beta-amyloid deposits in Alzheimer’s disease and alpha-synuclein buildup in Parkinson’s disease. It is unclear whether neurodegeneration in these conditions is due to protein aggregates or to other characteristic impairments in the sickly cells, including proteasome and mitochondrial dysfunction and the loss of trophic factors.

—Molly McElroy

Piece Added to Vertebrate Clockwork

A novel regulator in the mammalian circadian clock has been discovered in mouse, rat, and human tissues by HMS neurobiologists. The molecule, dubbed CLOCK-interacting protein, circadian (CIPC), helps turn off a transcription factor that positively drives the clock, thereby contributing to the inhibitory component of the circadian rhythm. The findings are reported in the March issue of Nature Cell Biology.

Charles Weitz, the Robert Henry Pfeiffer professor of neurobiology at HMS, previously reported that the main circadian gene CLOCK joined with the protein called brain muscle arnt-like-1 (BMAL1) to drive the circadian clock. Few molecular contributors to the clock were known in 1998, when Weitz reported the CLOCK–BMAL1 coupling in Science. Back then, Weitz and his team did screens to identify CLOCK’s partner, and BMAL1 jumped out from about 20 other proteins that emerged from their screen. “It’s a sequence expressed in muscle and brain, but that’s all people knew about it,” said Weitz of CLOCK’s in vivo partner.

Since that finding nearly 10 years ago, new evidence has hinted that other molecular players work to inhibit CLOCK–BMAL1. In their present work, Weitz and his research team took a fresh look at the 20-odd proteins that coexisted on the lab’s screens that partnered BMAL1 with CLOCK. Aided by more recent findings that clock mechanisms have been identified in many different body tissues, the researchers had a clearer idea of what to look for. Their haystack shrank further when they factored in the suspicion that the protein of interest might bind to a fragment near CLOCK’s C-terminus, a region that can alter clock function.

Passing these proteins through an additional screen, the researchers identified CIPC (“SIP-see”) as a negative regulator of the heterodimer CLOCK–BMAL1. CIPC showed circadian activity in each examined body tissue: liver, heart, and kidney. In each of these, CIPC was under CLOCK control.
Based on these in vitro findings, the researchers studied whether CIPC and CLOCK colocalized. Tissue slices stained for CIPC and CLOCK revealed the proteins’ nuclear cohabitation in all of the examined tissue types. Using a chicken antibody—when no other antibodies worked—the researchers found that CIPC and CLOCK coprecipitated. “That was a huge step,” Weitz said. “Having CIPC and CLOCK in a complex together takes it much further toward being reasonable that CIPC is part of a clock mechanism.”
The team also evaluated CIPC’s circadian contributions by depleting it through RNA interference. Knocking down CIPC shortened the clock’s cycle by an hour, which is considered a significant effect, Weitz said.

CIPC appears to be the first circadian rhythm protein without an invertebrate homologue, since the researchers did not find an equivalent protein in C. elegans or Drosophila. The ­vertebrate-specific protein lends support to the theory that mammalian clocks are more complex, though the behavioral consequences of having a more complex clock are unclear.

—Molly McElroy

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