06 Feb 2015

Because it goes hand-in-hand with neurodegenerative disease, researchers would like a reliable way to image inflammation in the brain. They have used PET tracers that target the mitochondrial translocator protein. TSPO ramps up in microglia when they turn on in response to inflammation. However, some people carry a polymorphism in the receptor that weakens binding. In the January 30 Science, two independent groups each reported a detailed structure of TSPO. Although each crystallized the protein from a different bacterium, their findings largely agree. The data show how the polymorphism alters the PET ligand binding site, which the authors believe will allow radiologists to develop more effective imaging agents. The researchers, based in New York City and Michigan State University, East Lansing, also examined the binding of endogenous ligands to TSPO, unearthing clues to potential roles for the receptor in hormone synthesis and protection from oxidative stress.

“These two articles provide crucial information on the structure and function of TSPO,” Michelle James at Stanford University, Palo Alto, California, wrote to Alzforum. She noted that elucidating how the polymorphism changes the protein’s shape was particularly helpful, but that more work will be needed to fully understand why various PET ligands bind with different affinities to the two versions of the protein.

TSPO sits in the outer mitochondrial membrane and is believed to ferry cholesterol and other molecules into these organelles. The protein marks activated, harmful microglia in the brains of people with Alzheimer’s and other neurodegenerative diseases (see Apr 2011 conference news). For decades, radiologists have visualized neuroinflammation using the PET ligand PK11195, which binds the transporter. However, this tracer has low sensitivity and specificity and poorly enters the brain. Several groups are developing alternative ligands that appear more selective (see Apr 2012 news; Mar 2013 conference news; Dec 2014 conference news). These newer agents bind poorly to TSPO that contains an alanine to threonine polymorphism at residue 147 (A147T). 

Detailed knowledge of the receptor’s structure would help chemists improve ligand design, but TSPO has resisted previous attempts to crystallize it. The research group led by Shelagh Ferguson-Miller at MSU succeeded by putting Rhodobacter sphaeroides TSPO in a fatty medium similar to the membrane it normally inhabits. X-ray analysis of the crystals indicated the receptor contained five transmembrane helices, with cholesterol binding in a groove between helices II and V. First author Fei Li introduced a mutation that mimics the A147T polymorphism into the bacterial protein, then crystallized that as well. The polymorphism, which occurs close to the cholesterol binding site, caused the helices to tilt and twist with respect to each other, narrowing the groove. This likely weakens the binding and transport of cholesterol, the authors note. Because mitochondrial cholesterol kicks off the synthesis of steroid hormones, the data may explain why people with the A147T polymorphism make less of them, the authors suggest (see Costa et al., 2009), and in turn are more susceptible to anxiety disorders affected by those hormones (see Nakamura et al., 2006; Costa et al., 2009; Colasanti et al., 2013).

How does the A147T polymorphism affect binding of PET tracers? Researchers led by Wayne Hendrickson at Columbia University crystallized Bacillus cereus TSPO in a complex with PK11195. First author Youzhong Guo identified a binding pocket between helices I and II, where the ligand nestled. This is not the site that binds cholesterol. While this group did not crystallize A147T variant TSPO, computer modeling predicted that the polymorphism would cause an alanine side chain to stick out into the pocket in a way that would likely block access.

TSPO also binds porphyrins, organic molecules that grab onto metal ions such as iron and assemble to form hemoglobin. Hendrickson and colleagues found that protoporphyrin IX settled into the same binding pocket as PK11195. Once there, TSPO catalyzed the degradation of this porphyrin, turning it from red to a deep blue compound that the authors named bilindigin. This previously unrecognized molecule resembles the blood-breakdown product biliverdin, which gives bruises their dark color. Biliverdin blocks the production of reactive oxygen species, suggesting that bilindigin may also play a role in protecting cells from oxidative stress. This could explain why microglia overproduce TSPO in response to inflammation and cellular stress, Hendrickson speculated. Other studies have reported that reactive oxygen species accumulate in TSPO knockouts (see Frank et al., 2007).

How closely do these bacterial proteins resemble the human transporter? The protein is highly conserved, particularly in the PK11195 binding pocket, with about one-third of its total residues identical between bacterial and human homologs. Rat TSPO can rescue bacteria that lack a copy of the TSPO gene, suggesting that function is conserved from prokaryotes to mammals. However, a recent study that described the structure of mouse TSPO using nuclear magnetic resonance imaging reported significantly different rotations of its transmembrane helices compared to the new bacterial data (see Jaremko et al., 2014).  Ferguson-Miller plans to crystallize human and mouse TSPO and assess how the polymorphism changes ligand binding in these molecules. The data would have implications not just for PET imaging, but also for understanding different disease states, Ferguson-Miller noted.—Madolyn Bowman Rogers

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References

News Citations

1. Barcelona: Inflammation—That Two-Faced Beast 19 Apr 2011
2. Imaging Inflammation: Can Glial PET Tracers Make a Mark? 27 Apr 2012
3. Glial Imaging—Amid Slow Progress, EU Project Takes Up Challenge 26 Mar 2013
4. Meet GE180: A PET Ligand for Tracking Neuroinflammation? 10 Dec 2014

Paper Citations

1. Costa B, Pini S, Gabelloni P, Da Pozzo E, Abelli M, Lari L, Preve M, Lucacchini A, Cassano GB, Martini C. The spontaneous Ala147Thr amino acid substitution within the translocator protein influences pregnenolone production in lymphomonocytes of healthy individuals. Endocrinology. 2009 Dec;150(12):5438-45. Epub 2009 Oct 21 PubMed.
2. Nakamura K, Yamada K, Iwayama Y, Toyota T, Furukawa A, Takimoto T, Terayama H, Iwahashi K, Takei N, Minabe Y, Sekine Y, Suzuki K, Iwata Y, Pillai A, Nakamoto Y, Ikeda K, Yoshii M, Fukunishi I, Yoshikawa T, Mori N. Evidence that variation in the peripheral benzodiazepine receptor (PBR) gene influences susceptibility to panic disorder. Am J Med Genet B Neuropsychiatr Genet. 2006 Apr 5;141B(3):222-6. PubMed.
3. Costa B, Pini S, Martini C, Abelli M, Gabelloni P, Landi S, Muti M, Gesi C, Lari L, Cardini A, Galderisi S, Mucci A, Lucacchini A, Cassano GB. Ala147Thr substitution in translocator protein is associated with adult separation anxiety in patients with depression. Psychiatr Genet. 2009 Apr;19(2):110-1. PubMed.
4. Colasanti A, Owen DR, Grozeva D, Rabiner EA, Matthews PM, Craddock N, Young AH. Bipolar Disorder is associated with the rs6971 polymorphism in the gene encoding 18 kDa Translocator Protein (TSPO). Psychoneuroendocrinology. 2013 Nov;38(11):2826-9. Epub 2013 Aug 12 PubMed.
5. Frank W, Baar KM, Qudeimat E, Woriedh M, Alawady A, Ratnadewi D, Gremillon L, Grimm B, Reski R. A mitochondrial protein homologous to the mammalian peripheral-type benzodiazepine receptor is essential for stress adaptation in plants. Plant J. 2007 Sep;51(6):1004-18. Epub 2007 Jul 25 PubMed.
6. Jaremko L, Jaremko M, Giller K, Becker S, Zweckstetter M. Structure of the mitochondrial translocator protein in complex with a diagnostic ligand. Science. 2014 Mar 21;343(6177):1363-6. PubMed.

Further Reading

News

1. Innate Immune Cells Enlisted to Clear Amyloid, Fight Disease 5 Dec 2013
2. Curbing Innate Immunity Boosts Synapses, Cognition 8 Aug 2013
3. Do Microglia in the Hypothalamus Drive Aging? 3 May 2013
4. Microglia Activation—Venusberg Meeting Questions M1, M2 Designations 14 Mar 2013
5. Soothing Neuroinflammation Quells Plaques in Mice 29 Nov 2012