Alzheimer’s-linked Protein Shows Unexpected Action

In biology, structural and functional similarities often go hand in hand. For this reason, it has been postulated that the amyloid precursor protein (APP) and the signaling molecule Notch, both cleaved by the same intramembrane protease, may work in a similar manner. Because Notch cleavage releases a cytoplasmic domain that translocates to the nucleus, activating gene transcription, this mechanism of action has been the paradigm for APP.

But in a dramatic twist to the evolving APP story, work from the lab of Bruce Yankner, HMS professor of neurology at Children’s Hospital Boston, suggests that release of the APP intracellular domain, or AICD, is not required for APP to signal to the nucleus and activate gene transcription. The finding could be a milestone because although this protein is best known for releasing the amyloid-beta peptide that forms amyloid plaques in those with Alzheimer’s disease, the biological function of APP has remained a mystery.

Courtesy Bruce Yankner

An alternative view of APP signaling. Contrary to previous predictions, the amyloid precursor protein, or APP, may regulate gene transcription without ever entering the nucleus. At the cell membrane, and together with another protein called Fe65, APP stimulates phosphorylation of the transcription factor Tip60 by cyclin-dependent kinases (CDK). Once phosphorylated, Tip60 enters the nucleus with Fe65, where they activate transcription.

Writing in the Nov. 4 Journal of Biological Chemistry, Yankner and Matthew Hass, an HMS graduate student in neuroscience, report that in cells devoid of presenilins—the proteases that release the AICD—APP still activates transcription. Hass discovered that it does so by binding to a transcription factor called Tip60. While Tip60 and the intracellular domain of APP have been shown to interact before, Hass demonstrates that they do so at the cell membrane, not inside the nucleus where Notch intracellular domains operate. This occurred not only in cultured cells, but also in the brains of normal and APP-transgenic mice.

So how might this APP–Tip60 interaction influence gene activity? Knowing that Tip60 can be modified by phosphorylation, the researchers wondered if this might be part of a signal transduction mechanism. In a series of experiments, Hass found that phosphorylation of Tip60 is, indeed, essential for APP to activate transcription and that this phosphorylation is brought about by cyclin-dependent kinases. “The data suggests that APP works in a classic kinase signaling cascade rather than in a Notch-like signaling pathway,” explained Yankner, who emphasized that the biological role of APP is still not well understood. As for the relevance of this work to disease, it suggests that mutations in APP that cause familial forms of Alzheimer’s could pack a double punch, causing more amyloid-beta to be released into the extracellular space and perturbing normal intracellular signaling events.

—Tom Fagan

Buckyballs Become Weapon Against Infection

Over the last 50 years, we have poked holes in bacteria with penicillin and nuked them with Neosporin. But the bugs are fighting back. With flesh-eating bacteria, drug-resistant Staphylococcus and Streptococcus, and biofilms, one could be forgiven for thinking that a few more bacterial mutations will whisk us back to the pre-penicillin days. Thankfully, there are some novel antimicrobial agents that might help in the war against infection. The latest weapon is not a gun or a bomb, but a buckyball.

In the October issue of Chemistry and Biology, Michael Hamblin, HMS associate professor of dermatology at Massachusetts General Hospital, and colleagues report that derivatives of these 60-carbon, soccer ball–shaped buckminsterfullerenes are effective and selective antimicrobials. What’s more, they are only active under visible light, making them perfect for photodynamic therapy (PDT), a technique whereby photons are used to activate an otherwise inert bacteriocide.

Buckyballs are ideally suited to PDT because their condensed aromatic rings absorb plenty of light, leading to excitation of electrons. That energy can be passed on to nearby oxygen molecules to generate reactive oxygen species, such as superoxide or singlet oxygen, that are toxic to bacteria. The major hurdle in using fullerenes in PDT has been getting them close to the target microbes.

Chemists Tim Wharton and colleagues at Lynntech Inc. in College Station, Texas, got around this problem by giving the fullerenes a positive charge. First author George Tegos, a research fellow in dermatology at MGH, and colleagues tested several of these cationic derivatives in cultures of Staphylococcus aureus and found that they both bound to the bacteria in the dark and killed them in the light. Under relatively low doses of white light, the best compounds eliminated all but 0.001 percent of the bacteria. The compounds were also effective against Escherichia coli, Candida albicans, and Pseudomonas aeruginosa. In tests against human fibroblasts, the compounds did kill some of the cells, suggesting that safety is a concern, but they still outperformed toluidine blue O, the only antimicrobial PDT compound currently used in a clinical setting.

“Part of the appeal of these molecules is that they penetrate human cells very poorly,” said Hamblin. That plus the ability to temporally and spatially activate the compounds makes them particularly attractive for safely treating local infections such as those in wounds, burns, skin, or
mucous membranes.

—Tom Fagan

Eating and Energy Expenditure Governed by Different Neuronal Pathways

With the flip of a switch, scientists have made significant progress toward illuminating the elusive brain circuitry that controls body weight. Using a genetic approach to turn genes on or off within distinct subsets of neurons in mice, a team led by Joel Elmquist and Bradford Lowell, HMS associate professors of medicine at Beth Israel Deaconess Medical Center, pinpointed a group of cells that directs food intake. Their study reveals that eating and calorie burning—factors that respond to the same chemical signals—are controlled by anatomically distinct neuronal pathways. The findings, published in the Nov. 4 Cell, may guide the future discovery of new therapeutic targets for obesity.

It is clear that the brain is the mastermind behind the waistline. It monitors many parameters throughout the body, including hormones and digested food in the bloodstream and neuronal signals from the gut. Then it sends messages to adjust caloric intake and metabolism appropriately, fending off the extra pounds that might otherwise accumulate. One of the key members in this relay is the melanocortin-4 receptor (MC4R), which regulates both food consumption and energy expenditure and, if mutated, causes severe obesity in mice and humans. But within the brain, MC4R is distributed across vast regions, leaving scientists substantial room to wonder which locations are important for keeping body fat in check.

To tackle this issue, Elmquist, Lowell, and their colleagues exploited the Cre/loxP system, a technique used in mice to manipulate gene expression within select anatomical areas. First, they engineered an inactive Mc4r allele by interrupting the gene with a stop sequence. Posting loxP sites, which are homing signals for Cre recombinase, on either side of this genetic stop sign gave the researchers the option of removing it to restore function to the crippled gene. By delivering Cre recombinase to the paraventricular nucleus of the hypothalamus (PVH)—a site that has been implicated in obesity and normally expresses high levels of the receptor—Elmquist and Lowell’s team reestablished MC4R activity only at this specific address in the brain. When compared to counterparts lacking all MC4R function, the resulting animals were markedly less obese due to restoration of normal levels of food intake. Their metabolism, however, remained sluggish. “That the PVH can regulate all of the -melanocortin--mediated food intake, but doesn’t touch energy expenditure–-regulation was really very surprising,” said lead author Nina Balthasar, an HMS instructor in medicine at BID.

Now, with all eyes on the PVH in its command over food consumption, the challenge before Elmquist and Lowell is to identify the specific neurons responsible for the task. Likewise, they hope to locate the nerves lying outside of the PVH that regulate energy output. “The end result of this is going to be a wiring diagram that you can put in a textbook and say, ‘This is how the brain controls body weight,’” Lowell predicts.

—Nicole Davis