David Riddell

ApoE Promotes the Proteolytic Degradation of Aβ

Apolipoprotein E is associated with age-related risk for Alzheimers disease and plays critical roles in Abeta homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Abeta from the brain. The endolytic degradation of Abeta peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Abeta degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Abeta load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Abeta from the brain and suggest that LXR agonists may represent a novel therapy for AD.

The LXR agonist TO901317 selectively lowers hippocampal Aβ42 and improves memory in the Tg2576 mouse model of Alzheimer's disease

Recent studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein to Abeta peptide. Moreover, cholesterol-rich apoE-containing lipoproteins may also promote Abeta clearance. Agonists of the liver X receptor (LXR) transcriptionally induce genes involved in intracellular lipid efflux and transport, including apoE. Thus, LXR agonists have the potential to both inhibit APP processing and promote Abeta clearance. Here we show that LXR agonist, TO901317, increased hippocampal ABCA1 and apoE and decreased Abeta42 levels in APP transgenic mice. TO901317 had no significant effects on levels of Abeta40, full length APP, or the APP processing products. Next, we examined the effects of TO901317 in the contextual fear conditioning paradigm; TO901317 completely reversed the contextual memory deficit in these mice. These data demonstrate that LXR agonists do not directly inhibit APP processing but rather facilitate the clearance of Abeta42 and may represent a novel therapeutic approach to Alzheimers disease.

Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeability.

The identification of small molecule aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligands reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..

(S)-N-(5-Chlorothiophene-2-sulfonyl)-β,β-diethylalaninol a Notch-1-sparing γ-secretase inhibitor

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimers disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.

BACE1 is not the rate-limiting enzyme in Aβ production in the brains of APPswe transgenic mice: Pharmacological characterization of BACE1 using novel brain penetrant inhibitors.

Background: Protein-protein interactions are a principal regulatory mechanism in biology, but also a common source of pathogenesis. Many proteins are susceptible to misfolding and amyloidogenesis, leading to human disease via a gain of pathogenic function. The assembly of these misfolded proteins into a variety of aggregates is linked to numerous debilitating diseases such as neurodegenerative diseases, polyneuropathies and cardiomyopathies. Recent studies from three diverse disciplines, neuropathology, genetics, and biophysics, have indicated a novel target for therapeutic intervention: ordered protein aggregation. However, previous small molecule based approaches to inhibit protein-protein interactions, particularly aggregating proteins, have had limited success, and subsequently inhibition of protein-protein interactions remains the Holy Grail of drug design. Methods: A few years ago we developed a new strategy that combines emerging chemical and biological technologies to address this problem. This strategy enables small molecules to recruit the steric bulk provided by the chaperone FKBP to the aggregating peptide and to thus effectively block the formation of pathogenic protein aggregates. Results: We have now expanded on this approach and have designed bifunctional small molecules that are able to recruit other cellular proteins that have been shown to modulate A-beta aggregation and to genetically interact with the A-beta aggregation pathway. To identify novel compounds that bind to different aggregation species of A-beta, we have used both rational design as well as an unbiased high throughput screening (HTS) approach, which we complement with computational scaffold hopping to identify structural analogs of hits. Conclusions: In addition to their potential as a starting point for the development of novel therapeutic agents for AD, these bifunctional small molecules can also be used as chemical probes to address unanswered questions regarding the molecular mechanisms underlying the pathogenesis of AD.

Examining the role of ApoE in LXR-Mediated Aβ clearance mechanisms: Implications for the development of Alzheimer's disease therapeutics

addition, we measured markedly elevated CSF aSyn levels (300.0 þ/ 248 pg/ul) in 14-3-3-positive Creutzfeldt-Jakob disease (CJD) patients that significantly differed from all other groups (CJD versus NCO, p < 0.001). Using the same technique, we also measured the concentration of total aSyn in serum and plasma from healthy subjects, which measured 15.0 þ/ 0.9 and 45.0 þ/ 1.4 pg / ul, respectively. Conclusions: Our data confirm the previous finding that aSyn is present in adult human CSF. In a cross-sectional study of 67 donors with neurodegeneration and 13 NCO subjects, we recorded a pathological rise of CSF aSyn in all definite CJD cases and observed the lowest mean concentration in our donors with advanced PD. Mean CSF aSyn concentrations were comparable among the AD and NCO groups, whereas the values for probable and definite DLB cases ranged between those for AD and PD. Ongoing studies seek to identify the source(s) and variants of CSF aSyn in living donors, and to further examine its utility in the diagnosis of neurodegenerative syndromes.

Implementing CompacT SelecT Automated Cell Culture in Support of Alzheimer's Disease-Targeted Drug Discovery

The CompacT SelecT is the latest generation automated cell culture system in the SelecT product line allowing incubation of up to 90 T-175 flasks and preparation of 210 assay-ready plates. We have successfully implemented the CompacT SelecT in support of a number of cell-based assays used in our Alzheimers disease (AD) lead optimization programs. One of the distinguishing features of AD pathology is deposition of two neurotoxic forms of the beta-amyloid peptide (Aβ40 and Aβ42) in the brains of patients. It is thought that specifically lowering Aβ40 and Aβ42 in the brains of patients will halt the progression of the disease. The generation of Aβ requires sequential cleavage of the type-I integral membrane amyloid precursor protein (APP) by two proteases, β-secretase (BACE) then γ-secretase. In the specific examples presented here, we have transitioned two cell lines supporting drug discovery efforts for identifying β- and γ-secretase inhibitors (GSIs) from manual cell culture protocols to fully automation using CompacT SelecT. In Chinese hamster ovary (CHO) cells which over express wild-type APP (CHOAPP cells), robust secretion of Aβ40 was observed from cells cultured manually and with CompacT SelecT with signal:background ratios of 54–99 and 23–47, respectively. Despite the reduced signal:background observed with the CompacT SelecT cultured cells, the rank order of potency for a series of 18 BACE inhibitors in reducing Aβ40 secretion was identical when manually cultured cells were compared with CompacT SelecT cultured cells. The correlation coefficient when comparing the two sets of EC50 values was r 2 = 0.99. Similarly, CHO cells that over express a reporter construct for the Swedish mutant APP that generates high levels of Aβ peptide (CHOAPP-NL cells) cultured with CompacT SelecT exhibited a reduced secretion of Aβ40 and Aβ42 when compared with manually cultured cells; signal:background ratios for secretion of Aβ40 from CHOAPP-NL cells were 57–71 and 16–31, and for Aβ42 secretion 8–18 and 4–11 comparing manual and automated cell culture, respectively. Similar to the observations in CHOAPP cells, an identical rank order of potency for a series of GSIs was observed when CHOAPP-NL cells cultured manually and in CompacT SelecT were compared; r 2 = 0.99 and 0.98, respectively, for EC50 values for inhibition of Aβ40 and Aβ42 secretion. Taken together, the robust signal:background and identical pharmacological profile of inhibitors provide validation for the implementation of automated cell culture for CHOAPP and CHOAPP-NL cells in support of β- and γ-secretase-targeted drug discovery.

P2-306: Activation of both LXRα and LXRβ is required for TO901317-mediated lowering of Aβ42: implications for the development of Alzheimer's therapeutics

sive agents decrease Alzheimer’s disease (AD) incidence. Previous study in our lab found that treatment with the angiotensin II receptor antagonist antihypertensive agent, valsartan, significantly attenuated spatial memory deterioration in the Tg2576 mouse model of Alzheimer’s disease (AD) in the absence of detectable changes in blood pressure. Moreover, we found that cognitive benefits of valsartan treatment coincided with significantly reduced accumulations high molecular weight (HMW)-soluble A species in the brain (Wang et al., 2007). Methods: Based on this evidence and the fact that HMW-soluble oligomeric A species in the brain are highly involved in the development of AD type cognitive deterioration, we continue to explore potential anti-A oligomerization activity among 55 antihypertensive drugs representative of all drug classes prescribed for hypertension. Results: In addition to valsartan, we used a cell-free assay and identified four drugs, namely furosemide (a diuretic), nitrendipine (a Ca channel receptor blocker), candesartan cilextil (an angiotensin II receptor antagonist), and diazoxide (a vasodilator) that exert anti-A oligomerization activity and significantly attenuate oligomerization of synthetic A 1-42 peptide into HMWsoluble A species in a dose-dependent manner (5-200 M). These four identified antihypertensive drugs showed no detectable A lowering activities in primary cortico-hippocampal neuron cultures derived from Tg2576 mouse embryos. Conclusions: Our study suggests, for the first time, that certain antihypertensive drugs might selectively protect against AD-type neuropathology through mechanism that inhibits oligomerization of A into HMW soluble species. Ongoing studies in the lab using Tg2576 AD mouse model will continue to test the potential roles of A -oligomerization-inhibiting antihypertensive drugs in AD prevention and/or therapies. Altschul Foundation, NIA AG02219, Neurological Disease Research Center FoundationReina Sofia Foundation, Dana Foundation for Brain Research Initiative to GMP.

P1-055: Impaired induction of neuronal immediate-early gene expression correlates with contextual memory deficits in APP transgenic mice

Background: Transgenic mouse models with neuronal expression of human amyloid precursor protein (APP) develop a range of Alzheimer’s disease (AD)-like alterations, including deposition of A and age-dependent deficits in learning and memory. The most popular hypothesis proposes that soluble, oligomeric forms of A mediate the memory deficits observed in APP transgenic animals prior to the formation of plaques. However, very little is known about the underlying molecular mechanisms leading to A mediated memory dysfunction. Methods: It has been known for some time that normal memory processes are associated with altered gene expression. This makes unraveling the molecular basis of memory dysfunction in AD ideally suited to transcriptional profiling. To that end, we have begun to characterize the molecular changes underlying early memory impairments in the Tg2576 mouse model of AD by analyzing the gene changes associated with memory formation in the brains of both wild-type and cognitively impaired Tg2576 mice. Results: In wild-type mice the formation of a strong contextual fear-related memory was associated with a robust induction (2-4 fold) of known immediate early genes (IEGs): c-fos, Jun-b, Erg1 and Nurr77, as well as the effector gene, Arc/Arg3.1 in both the amygdala and hippocampus. A number of genes not previously associated with memory formation were also significantly upregulated. None of these genes were induced in the Tg2576 implying that A antagonizes memory formation upstream of IEG induction. Conclusions: These data, together with recent reports by others, suggest a scenario where soluble A species impair neuronal signaling in key mechanisms related to memory formation. Many of the genes identified to be differentially regulated are downstream of NMDA receptor signaling, indicating the importance of these pathways in A induced memory impairments. We are currently examining the effect of A -directed therapeutics on reversing the memory and IEG deficits in the Tg2576 mice.

P4-085: The role of liver X receptor (LXR) agonists in modulating Aβ levels in vitro and in vivo

Recent studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein (APP) to A peptide, the major constituent of senile plaques in Alzheimer’s disease (AD). As well as influencing A production, cholesterol, and in particular, cholesterol-rich apolipoprotein E (apoE) containing lipoproteins may also promote A clearance and degradation. Agonists of liver X receptor (LXR) transcriptionally induce a variety of genes involved in cellular lipid efflux and transport, including ATP binding cassette A1 (ABCA1) and apoE. As such, LXR agonists have the potential to both inhibit APP processing and promote A clearance. Here we show that LXR activation yielded inconsistent effects on A production in vitro. A range of tool LxR agonists either decreased, stimulated or had no effect on A 40 and A 42 secretion. In vivo, LXR agonist TO901317 dose dependently increased brain ABCA1 and apoE levels with a concomitant decrease in A 42 levels in APP transgenic (Tg2576) mice. Interestingly, TO901317 had no significant effects on levels of A 40, full length APP, or the APP processing products; sAPP , sAPP , and CTF . Since A 42 has been linked to memory failure in the Tg2576, we examined the effects of TO901317 in the contextual fear conditioning paradigm; TO901317 completely reversed the working memory deficit in these mice. Together these data demonstrate that LxR agonists do not directly inhibit APP processing but rather facilitate the clearance of A 42 and improve working memory.