Familial AD (FAD) is caused by mutations in a small subset of genes that control the generation of Aβ 
. Because of these findings, it is widely believed that Aβ is the causative factor in AD and the vast majority of the drug development effort for AD is focused upon Aβ or components of the Aβ biosynthetic pathway. While Aβ is clearly relevant, over 95% of all AD cases are not FAD and age is by far the greatest risk factor for the disease. It is therefore likely that the various pathological components of old age such as oxidative stress, the loss of trophic factors, reduced energy metabolism, and inflammation all lower the threshold for Aβ toxicity. It follows that conditions that reduce these stressors are likely to delay the onset of AD and/or reduce its consequences 
. These age-associated parameters have not, however, been employed as a group for targeting pathways in AD drug discovery.
To explore the possibility of using these parameters for the discovery and development of AD drugs, we initially synthesized a library of hybrid molecules between the multi-target plant polyphenolic curcumin and a neurotrophic molecule, cyclohexyl-bisphenol A. We then selected a compound called CNB-001 that was neuroprotective in six toxicity assays 
. Selection was followed by a limited structure-activity (SAR) analysis of CNB-001, and a chemical reaction that was able to generate extensive diversity around the structural components of CNB-001 that the SAR suggested were required for activity. From the products of this reaction, we identified one compound, J147, that was active in the original assays but that had EC50
s up to 100 times lower than CNB-001 in the various selection assays. J147 also has greatly improved medicinal chemical properties for a CNS drug.
Since two of the six selection assays are directly for neurotrophic activity, and BDNF is a neurotrophic factor that is reduced in AD brain and is required for normal memory, we asked if J147 could modulate hippocampal LTP, memory, and prevent behavioral deficits in a huAPP/PS1 AD mouse model. shows that J147 is able to facilitate the induction of LTP in hippocampal slices. LTP requires a large number of signaling cascades, including those activated by BDNF 
. Like J147, exogenous BDNF promotes, but does not cause LTP 
. Conversely, LTP is attenuated in multiple strains of BDNF knockout mice 
. LTP is associated with the increased expression of BDNF and of the BDNF responsive gene, Homer 1. BDNF also increases the phosphorylation of PSD95. J147 mimics BDNF by both maintaining Homer 1 expression and increasing the phosphorylation of PSD95 in AD transgenic mice (). Because J147 has both long- and short-term biological effects, as well as in multiple cell culture aging/disease models, it is likely that efficacy is the result of pleiotrophic mechanisms. For example, BDNF-like neuroprotective activity does not require the expression of the BDNF receptor (), but it is able to induce BDNF expression in vivo
. In addition, the in vivo
metabolites of J147 have the same biological activities as the parent, which may contribute to its long-term efficacy (in preparation).
The normal aging process is often associated with a reduction in learning and memory, so it would be very useful for an AD drug to also enhance non-pathological memory loss. Several different types of memory tests that assess aspects of memory in humans can be performed in rodents 
. Working memory is assessed using the Y-maze 
, spatial memory using the Morris water and Barnes mazes 
, and recognition memory by using the novel object recognition test 
. J147 has broad cognitive enhancing effects, including enhanced working memory, object recognition and spatial memory in healthy adult rodents (). J147 also prevented the spatial learning and memory deficits observed in AD transgenic mice (). While there are many cognitive enhancing drugs that regulate aspects of synaptic transmission 
, J147 is a novel candidate, for it is neither a phosphodiesterase nor an acetylcholinesterase inhibitor.
There is an extensive literature showing that the BDNF signaling pathway is a major component of learning and memory 
. BDNF is highly expressed in the hippocampus 
, and cognitive enhancement paradigms such as exercise enhance pro-BDNF, BDNF, and BDNF mRNA in the hippocampus 
. Recently, it has been argued that individuals who get regular exercise are less prone to AD 
. Conditional BDNF gene deletion in adult mice causes impaired learning and memory in multiple assays, confirming its role in cognition 
. In addition, BDNF may enhance synaptic efficacy by promoting synaptic spine growth and stability, including the growth of new spines that increase synaptic density along neurites 
. J147 maintains the expression of synaptophysin, a synaptic vesicle protein that is reduced in both aging and AD, and is considered an excellent marker for synapse loss () 
. J147 also prevents the loss of drebrin and synapsin-1 in the huAPP/PS1 animals (). Drebrin is an actin binding protein that is enriched in synaptic spines that also declines with age and AD 
and synapsin-1 is a membrane protein that is involved in synaptic vessel release 
. These data show that J147 not only has the ability to enhance memory in both normal and AD mice, but to increase the expression of the protein substrates for memory.
Both oxidative stress and inflammatory markers are enhanced in human AD brain 
as well as in AD transgenic mice 
. The interpretation of biochemical markers for oxidative stress in the AD brain is, however, somewhat complicated because there are two populations of cells at the extremes of viability, one expressing the genes associated with a survival response and another reflecting activated cell death pathways. In the case of inflammation, a modest activity may be neuroprotective, for example amyloid phagocytosis by activated microglia or macrophages, while a more robust inflammatory response will be destructive. Therefore, from a therapeutic point of view, it is best to return the expression of the proteins involved to the pre-AD control level.
We examined four indicators of oxidative stress and inflammation in control, huAPP/PS1 and J147-treated huAPP/PS1 mice. Heme oxygenase 1 (HO-1) is elevated in AD 
, and in the AD transgenic animals (); J147 reduces expression to below control levels. Iba-1, a marker for microglia is significantly elevated in AD mice and reduced by J147, while a toxic microglial enzyme, iNOS, is not quite significantly reduced by J147 (). Finally, 5 LOX is a potent reactive oxygen species (ROS) producing and pro-inflammatory enzyme that is increased in AD 
. This enzyme is also increased in the transgenic mice but levels are dramatically reduced by J147 (). Together these data show that J147 is able to normalize several aspects of the pro-inflammatory and pro-oxidant conditions in the transgenic animals.
Finally, AD can be viewed as a protein misfolding disease that leads to the aggregation of many proteins, including Aβ, resulting in toxicity and, ultimately, cell death. Under conditions of stress, heat shock proteins 70 and 90 are induced to enhance protein clearance. HOP-1 (also called stress-inducible protein 1) is a co-chaperone that interacts with HSP70 and HSP90 families. HOP-1 plays crucial roles in the productive folding of substrate proteins by controlling the chaperone activities of HSP70 and HSP90. show that all three proteins are elevated in huAPP/PS1 mice and that J147 reduces their expression. The ability of J147 to lower the expression of heat shock proteins in AD transgenic mice again suggests that this unique compound has the ability to normalize multiple aspects of AD pathology.
The above data demonstrate the potential of a new drug discovery paradigm for AD that is based upon the requirement that drug candidates be highly effective in multiple, distinct cell culture models of neurodegeneration. This approach yielded a very potent, orally active drug candidate that targets several different pathways that decline in AD. A large number of potential pharmaceutical, nutritional, and immunological therapies have been tested in clinical trials for AD, but to date they have not altered cognitive decline in humans 
. In contrast, over 200 compounds appear to reduce plaque loads or behavioral deficits in AD transgenic mice 
and several compounds increase the amount of BDNF in rodent brain 
. However, none of these compounds nor any of the drugs that are in clinical trials for AD have the combination of activities of J147 in cell culture and in animals (
). J147 is unique in its ability to enhance LTP, potentiate learning and memory in both normal and AD transgenic animals, and maintain synaptic proteins while at the same time reducing biochemical markers of inflammation and soluble Aβ levels in AD transgenic mice. Therefore, the range of biological activities of J147 relevant to human AD is also much more extensive than any of the compounds that have failed in clinical trials. In addition, J147 is very potent, has good medicinal chemical properties for a CNS drug 
, is apparently safe, and is orally active. Finally, unlike the current drugs approved for AD, J147 is neither an acetylcholine esterase inhibitor, an NMDA receptor antagonist, nor a phosphodiesterase inhibitor, yet it enhances cognition with a short-term treatment. Thus J147 is an exciting new compound with the potential to be an AD therapeutic by slowing disease progression through neuroprotection as well as providing immediate cognition benefits. These dual attributes improve the chances for success as a disease-modifying drug as well as in short-term AD clinical trials that use currently accepted approvable measures of outcome.