Warren D. Hirst

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.

Investigation of leucine-rich repeat kinase 2

Mutations in leucine‐rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson’s disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity, may lead to novel therapeutic strategies for treating Parkinson’s disease. In this study, highly purified, baculovirus‐expressed proteins have been used, for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2. Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time‐resolved fluorescence resonance energy transfer (TR‐FRET) assay format (LanthaScreen™) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR‐FRET assays to assess the role of clinical mutations affecting LRRK2’s kinase activity. Our results suggest that only the most prevalent clinical mutation, G2019S, results in a robust enhancement of kinase activity with LRRKtide as the substrate. This mutation also affects binding of ATP to LRRK2, with wild‐type binding being tighter (Km,app of 57 μm) than with the G2019S mutant (Km,app of 134 μm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson’s disease may be identified.

Inhibition of c-Jun kinase provides neuroprotection in a model of Alzheimer's disease.

The c-Jun N-terminal kinase (JNK) pathway potentially links together the three major pathological hallmarks of Alzheimers disease (AD): development of amyloid plaques, neurofibrillary tangles, and brain atrophy. As activation of the JNK pathway has been observed in amyloid models of AD in association with peri-plaque regions and neuritic dystrophy, as we confirm here for Tg2576/PS(M146L) transgenic mice, we directly tested whether JNK inhibition could provide neuroprotection in a novel brain slice model for amyloid precursor protein (APP)-induced neurodegeneration. We found that APP/amyloid beta (Abeta)-induced neurodegeneration is blocked by both small molecule and peptide inhibitors of JNK, and provide evidence that this neuroprotection occurs downstream of APP/Abeta production and processing. Our findings demonstrate that Abeta can induce neurodegeneration, at least in part, through the JNK pathway and suggest that inhibition of JNK may be of therapeutic utility in the treatment of AD.

Selective Phosphodiesterase (PDE)-4 Inhibitors A Novel Approach to Treating Memory Deficit?

Phosphodiesterase-4 (PDE4) belongs to an important family of proteins that regulates the intracellular level of cyclic adenosine monophosphate (cAMP). Several lines of evidence indicate that targeting PDE4 with selective inhibitors may offer novel strategies in the treatment of age-related memory impairment and Alzheimer’s disease. The rationale for such an approach stems from preclinical studies indicating that PDE4 inhibitors can counteract deficits in long-term memory caused by pharmacological agents, aging or overexpression of mutant forms of human amyloid precursor proteins. In addition to their pro-cognitive and pro-synaptic plasticity properties, PDE4 inhibitors are potent neuroprotective, neuroregenerative and antinflammatory agents. Based on the fact that Alzheimer’s disease is a progressive neurodegenerative disorder that is characterised by cognitive impairment, and that neuroinflammation is now recognised as a prominent feature in Alzheimer’s pathology, we have concluded that targeting PDE4 with selective inhibitors may offer a novel therapy aimed at slowing progression, prevention and, eventually, therapy of Alzheimer’s disease.

Sonic Hedgehog signaling in astrocytes is dependent on p38 mitogen-activated protein kinase and G-protein receptor kinase 2

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh‐regulated gene targets, Gli‐1 and Patched in a cyclopamine‐ and forskolin‐sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular‐signal‐regulated kinases or c‐jun N‐terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh‐dependent up‐regulation of Gli‐1, inter‐alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin‐like growth factor 2 or a novel Shh target, membrane‐associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively‐active mutant we show that Shh signaling to p38 MAPK and subsequent Gli‐1 transcription requires G‐protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G‐protein receptor kinase 2‐dependent p38 MAPK activity in regulating Shh‐mediated gene transcription in astrocytes.

Identification of pyridazino[4,5-b]indolizines as selective PDE4B inhibitors.

Substituted pyridazino[4,5-b]indolizines were identified as potent and selective PDE4B inhibitors. We describe the structure-activity relationships generated around an HTS hit that led to a series of compounds with low nanomolar affinity for PDE4B and high selectivity over the PDE4D subtype.

Chapter 3 Small-Molecule Protein–Protein Interaction Inhibitors as Therapeutic Agents for Neurodegenerative Diseases: Recent Progress and Future Directions

Publisher Summary This chapter highlights the recent progress in the development of small-molecule protein–protein interaction inhibitors that have applications in expanding the mechanistic understanding of neurodegenerative diseases, which can potentially lead to the development of rational therapeutics. Substantial genetic and physiological evidence suggest that the A β plays a central role in Alzheimers disease (AD) pathogenesis. A β is a 39-to 42-aminoacid peptide derived from the proteolytic processing of the amyloid precursor protein (APP) by secretases. Gradual changes in the steady-state levels of A β in the brain are thought to initiate the amyloid cascade. The chapter makes two general assumptions regarding the inhibition mechanism of amyloid protein fibril formation by small molecules: (1) specific structural conformation is necessary for β -sheet interaction and stabilization of the inhibition-protein complex and (2) aromatic interaction between the inhibitor molecule and the aromatic residues in the amyloidogenic sequence, potential “hotspots,” may direct the inhibitor to the amyloidogenic core blocking the protein–protein interaction. These assumptions are highly relevant for future design of small-molecule inhibitors as therapeutic agents for the treatment of amyloid-associated diseases.

Benzimidazole- and indole-substituted 1,3′-bipyrrolidine benzamides as histamine H3 receptor antagonists

Using a focused screen of biogenic amine compounds we identified a novel series of H(3)R antagonists. A preliminary SAR study led to reduction of MW while increasing binding affinity and potency. Optimization of the physical properties of the series led to (S)-6n, with improved brain to plasma exposure and efficacy in both water intake and novel object recognition models.

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.

Neuroprotective Agents for the Treatment of Ischemic and Hemorrhagic Stroke

Publisher Summary This chapter focuses on molecules and mechanisms that are at considerably earlier stages of development, are novel approaches to neuroprotection, and may become the next generation of drugs for the treatment of acute ischemic and hemorrhagic strokes. Neuroprotection by caspase inhibitors and poly [adenosine diphosphate (ADP)-ribose] polymerase inhibitors is described. Caspases are a family of cysteine proteases that are expressed as inactive zymogens and undergo proteolytic maturation in a sequential manner. Poly (ADP-ribose) polymerase-1 (PARP-1) is a chromatin-bound nuclear enzyme involved in a variety of physiological functions related to genomic repair, including deoxyribonucleic acid (DNA) replication and repair, cellular proliferation and differentiation, and apoptosis. Peroxisome proliferator-activated receptors (PPARs) are orphan receptors belonging to the steroids/thyroid/retinoid receptor super family of ligand-activated transcription factors. There are three peroxisome-proliferator-activated receptor (PPAR) isoforms (PPAR-α, -β, -γ), each of which is differentially expressed and displays a distinct pattern of ligand specificity. Src-family protein tyrosine kinases (SFKs) are important signaling enzymes controlling cell growth, proliferation, and migration.