TARGETING THE CREB PATHWAY FOR MEMORY ENHANCERS

1. TARGETING THE CREB PATHWAY FOR MEMORY ENHANCERS Tim Tully*, Rusiko Bourtchouladze*, Rod Scott* and John Tallman* Helicon Therapeutics and Cold Spring Harbor Laboratory

2. How are potential molecular targets for drugs found? • How are potential memory drugs discovered and evaluated? • What memory-related diseases are good candidates for CREB-related therapy?

3. ACETYLCHOLINE, GLUTAMATE • the neurotransmitters acetylcholine and glutamate • CHOLINERGIC • Pertaining to acetylcholine neurotransmission. • CHOLINESTERASE • An enzyme that metabolizes the neurotransmitter acetylcholine. Cholinesterase inhibitors lead to increased levels of acetylcholine in the brain. • GLUTAMATERGIC • Pertaining to glutamate neurotransmission.

4. Alzheimer’s disease (AD) pathology • associated with a progressive loss of cholinergic neurons • historically the pharmaceutical industry has focused on the identification of cholinesterase inhibitors. • Marketed AD drugs such as donzepil, rivastigmine and galantamine yield mild improvements • effects are variable and not long-lasting

5. The biochemical basis of memory. • NMDA (N-methyl-D-aspartate)-receptor activation (in mammals) seems to be central to the local, transient biochemical response at the synapse. • These transient biochemical responses induce changes in gene expression in the cell nucleus via activation (that is, phosphorylation) of the transcription factor cAMP-response-element-binding protein (CREB). • This transcriptional response also depends on NMDA receptor activation.

6. Protein kinase A (PKA) and mitogen-activated protein (MAP) kinase signalling pathways play dominant roles in activation. • CREB regulates a transcription factor cascade ultimately involved in a growth process that yields synapse-specific structural changes. • This process seems to involve microtubule-dependent transport of nascent mRNAs to synaptic regions and local regulation of translation.

7. A molecular switch for LTM. • Neurogenetic studies have shown CREB to be a key control point for LTM formation. • Loss-of-function manipulations of CREB leave learning and STM intact, but impair LTM.

8. Gain-of-function manipulations leave learning and STM intact, but enhance LTM formation specifically by reducing the amount of training required to produce maximal LTM. • Similar manipulations of CREB also produce changes in synaptic structure and function in several animal models and in various regions of the mammalian brain.

9. Molecular targets for drug discovery are those, such as CREB, which act as molecular switches. • many genes are involved in stages of memory formation. • likely several genes will act as molecular switches and are potential targets for drug screening. • combinations of drugs may provide more effective treatments.

10. Two classes of drug targets • a-amino-3-hydroxy-5-methyl-4-isoxazole propionicacid (AMPA) receptors • genes in the CREB pathway

11. AMPA receptors depolarize postsynaptic membranes in response to glutamate • Modulators of AMPA receptors have been developed on the basis of the expectation that they might facilitate NMDA receptor-dependent induction of LTP and, thereby, the acquisition of new memories. • Several of these compounds have yielded encouraging results from preclinical animal testing and are now undergoing clinical evaluations.

12. CREB / cAMP genes • ADENYLYL CYCLASE (AC). • An enzyme that synthesizes cAMP from ATP and is involved in intracellular signalling, usually after neurotransmitter activation. • PHOSPHODIESTERASE (PDE). • An enzyme that hydrolyzes cAMP into AMP.

13. Screen for enhancers of CREB signalling. • molecular target was discovered to be PHOSPHODIESTERASE4 (PDE4).

14. Assay for CREB-based memory enhancement. • naive mouse is placed into a novel chamber containing distinct visual, olfactory and tactile cues. • mouse receives mild electroshock to its feet. • mouse will remember for sometime afterwards that the chamber is dangerous. • When placed back into the chamber at a later time, the mouse will freeze, sitting stone still for many seconds, which is its natural response to danger. • percentage of time during an observation period that the mouse spends frozen represents a quantitative measure of its memory of a dangerous place.

15. normal mouse will learn this task in just one training trial. • Tully modified procedure so that more training trials were required to form maximal LTM. • By reducing the intensity of footshock, four-day memory retention in normal mice (C57/Bl6) was near zero after one training trial and required five training trials to reach maximum levels.

16. In initial experiments with drugs, animals were cannulated and drug (or vehicle alone) was injected directly into the hippocampus shortly before training. • Mice received two (submaximal) or five (maximal) training trials. • A number of Helicon’s PDE4 inhibitors, and the prototypic PDE4 inhibitor rolipram, enhanced four-day memory after two training trials, but had no effect on memory after five training trials.

17. The drugs were then injected intraperitoneally shortly before training to test their ability to penetrate the blood–brain barrier. • A number of Helicon’s compounds and rolipram again enhanced four-day memory after two, but not five, training trials. • Stated another way, the PDE4 inhibitors enabled memory to form following less than half the normal amount of training.

18. Was this effect of the drug specific to one behavioural task, or did it also work with other stimuli and sensory modalities? • addressed by evaluating a second behavioural task — object recognition — that relies on a mouse’s natural exploratory behaviour.

19. during training for this task, mice are presented with two identical novel objects, which they then explore for some time by orienting toward, smelling and crawling over. • presented at a later time with two different objects, one of which was presented previously during training and thus is ‘familiar,’ and the other of which is novel.

20. Found that drug-injected mice formed maximal memory with less-than-normal training • Thus, PDE4 inhibitors yielded similar effects on memory formation in two distinctly different behavioural tasks, • Evidence that their mechanism of action was on the memory process per se rather than on a more nonspecific aspect of a particular behavioural response.

21. Further steps in drug development

22. The blood–brain barrier • This barrier comprises endothelial cells of brain capillaries. • The junctions between these cells are extraordinarily tight and prevent most classes of molecules from moving freely from the blood to the brain. • This protective function probably evolved to spare the brain from assault by toxic substances, viruses and peripheral hormones.

23. Unfortunately, it also prevents many classes of therapeutic drugs from easily entering the brain. • the blood–brain barrier is much reduced in some areas of the brain, such as the hypothalamus where hormonal signals are exchanged with the periphery

24. To circumvent the blood–brain barrier for normal metabolism, transport proteins have evolved that actively carry sugars, amino acids and vitamins into the brain. • Attempts have been made to use these transport proteins to carry drugs, such as glutamate-receptor agonists and antagonists, but the rate of transport has not been sufficient to deliver adequate doses of drug to the brain.

25. The classic way to penetrate the blood–brain barrier is to design small heterocyclic organic molecules. • These molecules are generally constructed to have a high degree of lipid solubility (that is, lipophilicity) and a relatively small size (< 500 daltons). • Drugs such as rolipram and Helicon’s compounds were designed with this in mind.

26. Such design constraints introduce other constraints, however, which can include poor solubility and high affinity for plasma proteins, such as albumin, leaving only a small amount of drug free to equilibrate across the membrane.

27. Target specificity (or lack thereof) and high affinity (< 10 nM) are also desirable, but, nonetheless, must be balanced against increased size and the attendant decrease in brain penetrance. • CNS drug design constitutes a series of compromises to balance these various drug properties.

28. The P-glycoprotein problem • Achieving the optimal design to penetrate the blood–brain barrier does not automatically ensure that therapeutic levels of drug will reach the brain. • There are powerful mechanisms in the more permeable areas of the brain, such as the choroid plexus, that actively pump noxious substances out of the brain. • One of these pumps, P-glycoprotein, was first recognized as the multi-drug resistance factor in cancer chemotherapy

29. Unique side effects for central nervous system drugs • Molecular targets for central nervous system (CNS) drugs are usually involved in normal neuronal functions. • Moreover, they often function in different types of neurons (excitatory versus inhibitory, for instance). • Consequently, CNS drugs can have side effects that are specific to various brain functions.

30. Excitotoxicity • Some CNS drugs can have excitatory effects on neuronal activity, which induce seizures. These may be a direct effect of the drug or result from indirect activation of excitatory transmitter (glutamate) release.

31. Depressant • CNS drugs can have inhibitory effects on neuronal function that promote drowsiness and even unconsciousness and death. These activities can result from membrane stabilizing (or local anaesthetic) actions or from interaction with inhibitory transmitters, such as GABA (-aminobutyric acid).

32. Emesis • CNS drugs with specific dopamine receptor activation can cause emesis and nausea.

33. Weight gain • Some nonselective antipsychotic medications, such as olanzepine, cause weight gain. • Receptor knockouts in rodents have suggested that the 5-HTreceptor may be involved.

34. Sexual dysfunction • Selective serotonin re-uptake inhibitors (SSRIs) are known to produce sexual dysfunction. • Sexual dysfunction has not been reported as frequently with other classes of re-uptake blocker (noradrenaline and dopamine) and with monoamine oxidase inhibitors. • All can function as antidepressants.

35. For what diseases might CREB-based memory enhancer work best?

36. Stroke rehabilitation • The extensive, repetitive training that stroke patients undergo after recovering from the acute ischaemic trauma is likely to invoke synaptic plasticity to ‘remodel’ neuronal connections surrounding the damaged region, thereby recovering a certain degree of lost function65–68. • Co-administration of a CREB-based memory enhancer and training might yield more rapid functional recovery (that is, less training will be required to regain performance).

37. Alzheimer’s disease • CREB dependent gene transcription apparently is not involved in the pathology (neurodegeneration) associated with AD. • Moreover, CREB-dependent memory formation involves the normal chemistry of healthy neurons. • Hence, the efficacy of CREB-dependent memory enhancers might be expected to be lowest when treating patients with moderate to severe AD.

38. Treatment of early-stage AD patients • Animal models of ß-amyloid pathology show that memory defects develop well before any signs of neurodegeneration. • As neurodegeneration proceeds, however, individual neurons die more or less randomly within particular brain regions. • A kind of mini-stroke.

39. Psychological studies of human amnesiacs, mnemonists and people with exceptional or normal memory, have demonstrated that memory is a distinctive cognitive function that can be measured and studied independently of other cognitive abilities that humans possess (for example, reasoning, planning, abstracting, sequencing, language) or brain functions such as perception, motivation, emotion or motor activities.

40. H.M. • The patient known as H.M., who has arguably become the single most studied and quoted patient in the history of medical research, developed a severe memory deficit following surgical removal of portions of his medial temporal lobes (including the hippocampus). • H.M.'s severe memory loss, however, was contrasted by intact cognitive functions such as perceptual learning, language and reasoning.