10 Jan 2011

New research in this week’s Journal of Neuroscience sustains hopes of boosting cognition through enhancement of the cyclic AMP (cAMP) signaling pathway that turns on memory genes. Using genetic and RNA interference-mediated knockdown approaches in mice, Han-Ting Zhang, West Virginia University Health Sciences Center, Morgantown, and colleagues have pinned cognitive benefits to inhibition of the PDE4D subtype of phosphodiesterase-4. This enzyme breaks down cAMP. What’s more, specifically curbing PDE4D variants in the hippocampus does not appear to cause vomiting (emesis), a side effect that has stymied development of PDE4 inhibitors for Alzheimer’s and other diseases.

“It's pretty exciting stuff,” Alex Burgin of Emerald BioStructures, Bainbridge Island, Washington, said of the new study. “They clearly show that PDE4D is important for memory, and that by going after specific isoforms, you can separate the benefit of memory from the side effect of emesis.” Burgin, working with Mark Gurney and others who were at deCODE genetics in Reykjavik, Iceland, designed several PDE4D partial inhibitors that improve memory in mice without triggering a surrogate measure for vomiting (Burgin et al., 2010 and ARF related news story).

Memory formation relies on expression of genes upregulated by the transcription factor CREB, which springs into action when phosphorylated by enzymes downstream of cAMP. Phosphodiesterase-4 tones down this pathway by keeping cAMP levels at bay, spurring scientists to try enhancing cognition by attenuating PDE4. However, because the enzyme comes in four subtypes (A-D) that get spliced at least 25 different ways, it’s been tough to figure out which ones to block.

The current study zeroes in on PDE4D. The work was led by first author Yun-Feng Li, who has moved to the Beijing Institute of Pharmacology and Toxicology in China. Li and colleagues went after PDE4D because of mouse studies revealing its predominant expression in the hippocampus, memory loss attributable to its overexpression (Giorgi et al., 2004), and enhanced hippocampal long-term potentiation due to PDE4D deficiency (Rutten et al., 2008). Strangely, though, PDE4D knockout (4DKO) mice with increased hippocampal LTP seemed to fare poorly in tests of fear-conditioning memory, suggesting a complex relationship between PDE4D and cognition.

To get a closer look at this connection, Zhang’s team put the 4DKO mice through three other behavioral tests, all measuring hippocampal-dependent memory. In the first test, the researchers starved knockout and wild-type mice for a day, then set them loose to find food stashed in several compartments of an eight-arm radial maze. Hungry animals learn quickly where the grub is, but the 4DKO group remembered even better—making fewer errors than did wild-type mice when hunting for food in subsequent trials. The phosphodiesterase-deficient mice also excelled in object recognition and Morris water maze tests.

The researchers also examined the effects of the PDE4 inhibitor rolipram, given by intraperitoneal injection. Rolipram was used years ago to treat depression, but it induced nausea and was only minimally effective. The compound is no longer given to people, but is widely used in animal studies, where it has been shown to activate CREB, induce hippocampal LTP, and enhance memory. In object recognition and water maze tests, the 4DKO mice fared comparably to rolipram-treated wild-type mice, and both groups outperformed untreated wild-type mice. Rolipram-treated 4DKO mice, however, got no further cognitive boost, consistent with the importance of the PDE4D isoform in mediating rolipram’s memory effects. By confocal microscopy, the researchers also showed that hippocampal neurons from 4DKO and rolipram-treated wild-type mice had increased levels of phosphorylated CREB, connecting the cognitive effects with the cAMP/CREB pathway.

These data support a scenario where PDE4D suppression improves memory by maintaining cyclic AMP, which in turn drives genes needed for long-term memory. Another set of experiments suggested that PDE4D inhibition also promotes hippocampal neurogenesis. Wholesale phosphodiesterase-4 inhibition has this effect (Nakagawa et al., 2002; Sasaki et al., 2007; Li et al., 2009), but the new data specifically point to the D isoform as an inhibitor of neurogenesis. For these studies, Zhang and colleagues injected vehicle- and rolipram-treated mice (wild-type and 4DKO) with bromodeoxyuridine (BrdU), which marks dividing cells. Knockout and rolipram-treated wild-type mice had more BrdU-positive neurons in the dentate gyrus, suggesting that PDE4D blocks neurogenesis. Rolipram treatment did not further increase BrdU counts or CREB phosphorylation in the knockout mice, establishing the PDE4D isoform as a prime neurogenesis blocker. It is not clear if this is related to memory.

PDE4 variants are further subdivided into four groups based on the presence or absence of two unique conserved regions (UCR1 and UCR2) at the N-terminus. There are 11 different PDE4D isoforms, seven of which contain both UCR1 and UCR2. To find which of these isoforms mediate memory, the scientists designed microRNAs (miRNAs) targeting part of the UCR1 domain. Carried on lentiviral vectors, the miRNAs were injected into the dentate gyrus, where they selectively decreased expression of PDE4D4 and 4D5, UCR1/2-containing 4D variants highly expressed in the hippocampus. Knockdown of 4D4 and 4D5 recapitulated the same sorts of benefits seen with 4DKO and rolipram-treated wild-type mice—increased CREB phosphorylation, enhanced neurogenesis, and better performance in the object recognition test and in another measure of long-term memory (step-down passive avoidance test).

Furthermore, specifically targeting the 4D4 and 4D5 variants seemed to avoid the signaling pathways that trigger vomiting. Rodents do not vomit, hence, researchers assess emetic responses using a surrogate measure—resistance to the anesthetic effects of xylazine and ketamine (Robichaud et al., 2002). The researchers found that in both 4DKO and rolipram-treated wild-type mice, anesthesia wore off more quickly, but in the UCR1 miRNA-treated mice, it did not. This suggested to the authors that knocking down PDE4D-4 and -5 in the hippocampus may avoid nausea-related side effects.

“The paper is very valuable in defining, of the many isoforms of PDE4, which ones are important in memory,” said Michael Shelanski of the Taub Institute at Columbia University in New York City. “It’s novel and well done, and could possibly allow for refining therapeutic approaches.”

That could be years away, though, as designing isoform-specific inhibitors is tricky, Shelanski said. He noted a study in which downregulation of a different PDE4D variant (4D3) promoted heart failure and arrhythmias (Lehnart et al., 2005).

Several companies have had their eye on PDE4 as a potential therapeutic target for cognitive enhancement, even for treating Alzheimer’s. In 2008, Merck completed a Phase 2 trial on a selective PDE4 inhibitor (MK-0952; see Gallant et al., 2010) in mild to moderate AD patients. Another PDE4 antagonist (HT-0712; see MacDonald et al., 2007) is being developed by San Diego-based Helicon Therapeutics, Inc. According to the company website, HT-0712 was licensed from Orexo, a Swedish pharmaceutical company, and tested in a 28-day Phase 2a study of seniors with age-associated memory impairment. Though safe and well tolerated, the compound was reported to have “no effects” on measures of short-term memory or passive EEG measures, and “a statistically significant effect at one dose on long-term memory of word list learning.” Helicon is currently recruiting for a small study using positron emission tomography (PET) imaging to explore relationships between HT-0712 dose and inhibition of brain PDE4 activity in order to optimize dosing for subsequent trials.

Roche jumped into the fray, too. The company completed a series of Phase 1 trials of MEM 1414, the lead candidate from now-defunct Memory Pharmaceuticals’ PDE4 inhibitor program. In those studies, the compound was “safe and generally well tolerated” by healthy elderly volunteers, according to an excerpt from Memory Pharmaceuticals’ 2008 annual report to the Securities and Exchange Commission. In 2005, Roche stopped clinical development of MEM 1414 and its back-up candidate, MEM 1917, and Memory Pharmaceuticals regained rights to these agents, intending to move them into Phase 2 trials. However, Roche acquired the biotech company in a 2008 merger, and it is unclear whether MEM 1414, or any of the other PDE4 inhibitors, are currently being developed.

And what about the PDE4D partial inhibitors recently reported by deCODE genetics? The U.S. Food and Drug Administration gave the go-ahead for Phase 1 testing, Burgin told ARF. But then deCODE went bankrupt and re-emerged in early 2010 with an exclusive focus on DNA-based diagnostics, leaving the PDE4D inhibitors “on the shelf,” said Gurney, lead investigator of the research on those compounds (Burgin et al., 2010). Gurney, who is now a biotech consultant and investor through Tetra Discovery Partners, LLC, in Grand Rapids, Michigan, told ARF he is not aware of any PDE4-targeting compounds in clinical testing at present, though “discovery-stage chemistry is ongoing at several companies.”

In Shelanski’s view, rolipram, the classic PDE4 inhibitor shunned for its emetic effects, might still have a fighting chance as a cognitive enhancer. Some cancer drugs also induce vomiting but have been scaled back to avoid these ill effects. The same could be possible for rolipram, Shelanski suggested. In prior studies he did in collaboration with Taub colleague Ottavio Arancio (Gong et al., 2004 and ARF related news story; Smith et al., 2009), rolipram rescued synaptic function and memory in AD transgenic mice, and the improvements persisted for months. That suggests the possibility of episodic treatment, Shelanski said. He said he has been unable to convince the Alzheimer’s Disease Cooperative Study (ADCS) to consider an AD trial of rolipram. “Nobody is interested because it’s out of patent,” Shelanski told ARF.

Meanwhile, the Zhang lab is following up its recent study by trying to further pinpoint which PDE4D variants are involved in memory. The group is analyzing potential contributions of other PDE4 isoforms. PDE4A, for example, is found at low levels in the two brain areas involved in emesis (area postrema and nucleus of solitary tract), and could make a better drug target than PDE4D, which is highly expressed in those regions, Zhang told ARF.

Zhang thinks the current paper does not prove that blocking PDE4D isoforms would bypass the vomiting effects typically induced by such compounds. “At this point, we cannot conclude that inhibiting D4 and D5 would avoid the emesis problem,” he told ARF, noting that the proteins were knocked down by injecting miRNAs directly into the hippocampus, a brain region not involved in emesis.

“The pharma industry has shied away from PDE4 inhibitors because of their emetic response,” Burgin said. “But I think our paper on PDE4D allosteric modulators, and this new paper, show you can clearly separate the memory benefits from the emetic side effects, and will hopefully have the industry re-evaluating that decision.”—Esther Landhuis