Ann-Cathrin Radesäter

First and Second Generation γ-Secretase Modulators (GSMs) Modulate Amyloid-β (Aβ) Peptide Production through Different Mechanisms

Background: γ-Secretase modulators (GSMs) hold potential as disease modifiers in Alzheimer disease; however, their mechanism of action is not completely understood. Results: Second generation in vivo active GSMs were described and shown to modulate Aβ production via a non-APP targeting mechanism, different from the NSAIDs class of GSMs. Conclusion: A growing class of second generation GSMs appears to target γ-secretase and displays a different mechanism of action compared with first generation GSMs. Significance: The identification of in vivo active non-APP targeting second generation GSMs may facilitate the development of novel therapeutics against AD. γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-β (Aβ) peptides. The Aβ42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aβ production by targeting the APP. Here, we describe novel GSMs that are selective for Aβ modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aβ both in cell and cell-free systems as well as lower amyloidogenic Aβ42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a 3H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aβ modulation and have a different mechanism of action compared with the original class of GSMs described.

AZ-4217: a high potency BACE inhibitor displaying acute central efficacy in different in vivo models and reduced amyloid deposition in Tg2576 mice.

Aβ, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimers disease (AD). β-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to Aβ peptides. Small molecule BACE1 inhibitors are expected to decrease Aβ-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pm in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of Aβ production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.

The vitamin-E analog trolox and the NMDA antagonist MK-801 protect pyramidal neurons in hippocampal slice cultures from IL-1β-induced neurodegeneration

The neurotoxic effect of the pro-inflammatory cytokine interleukin (IL)-1β was studied in monolayer cultures, obtained using roller-drum incubation of hippocampal slices from neonatal Sprague Dawley rats. Following exposure to recombinant rat IL-1β for four days, a concentration dependent loss was observed in the number of NMDAR1 receptor subunit immunoreactive pyramidal neurons in the cultures, reaching significance at 10 ng/ml rIL-1β. Also incubation with recombinant mouse IL-1β caused a loss of pyramidal neurons, with a significant effect at a concentration of 30 pg/ml. The vitamin E analog trolox (30 μM) was found to exert a protective effect against the rIL-1β-induced neuronal degeneration. A neuroprotective action against rIL-1β was also found after co-incubation with the NMDA antagonist dizocilpine (MK-801; 30 μM), while no protection was found with the GABAA mimetic clomethiazole. Hence, the proinflammatory cytokine IL-1β is neurotoxic to hippocampal pyramidal neurons when studied in anin vitro system with advanced phenotypic characteristics. The neuroprotective effects exerted by trolox and MK-801 suggest that free radicals and NMDA receptor-mediated processes are involved in IL-1β-induced neurodegeneration.

Salmonella lipopolysaccharide (LPS) mediated neurodegeneration in hippocampal slice cultures

Neuroinflammation has been suggested to play an integral role in the pathophysiology of various neurodegenerative diseases. Bacterial lipopolysaccharide (LPS) endotoxins are general activators of immune-cells, including microglial cells, which induce expression of pro-inflammatory factors. The aim of this study was to characterize neurodegenerative effects of exposure to LPS, derived from Salmonella abortus equi bacteria, in an in vitro brain slice culture system. Quasi-monolayer cultures were obtained using roller-drum incubations of hippocampal slices from neonatal Sprague Dawley rats for three weeks. Microglia/macrophages were identified in the monolayer cultures by CD11b immu-nostaining, while neuronal populations identified includedN-methyl-D-aspartate (NMDA-R1) receptor immunoreactive pyramidal neurons and smaller GABA-immunoreactive cells. Following exposure to LPS (100 ng/ml) an increased density of CD11b positive cells was found in the cultures. In addition, the LPS exposure produced a concentration-dependent loss of the NMDA-R1 immunoreactive neurons in the cultures which was substantial at 100 ng/ml LPS. The loss of NMDA-R1 cells was apparent already after 24 h exposure to LPS and seemed to be primarily due to necrotic-like cell death. However, a continued loss of cells was found when cultures were analyzed at 72 h, concomitant with an increase in the expression of p53 in the NMDA-R1 cells and TUNEL labeling of a few cells. Also the number of GABA-immunoreactive cells decreased rapidly and to a substantial extent after 24 h exposure to LPS, with a continued decrease up to 72 h. The findings show that Salmonella LPS increases the density of CD11b positive cells and acts as a potent neurotoxin in hippocampal roller-drum slice cultures. The LPS-induced neurodegeneration has both necrotic- and apoptotic-like properties and appears to be non-selective, affecting both pyramidal and GABA neurons. LPS-induced neurotoxicity in slice cultures may be a useful system to study processes involved in inflammatory-mediated neurodegeneration. Keywords: Lipopolysaccharide; Neuroinflammation; Microglia; Neurodegeneration; Pyramidal neurons; NMDA receptors; GABA neurons

UV-induced apoptosis in SH-SY5Y cells: Contribution to apoptosis by JNK signaling and cytochrome c

Activation of the c‐Jun N‐terminal kinase (JNK) pathway is suggested to be required for neuronal apoptosis. We investigated the role of JNK on phosphorylation of c‐Jun, Bcl‐2, and apoptotic translocation of cytochrome c (cyt c) in UV‐induced apoptosis in human neuroblastoma SH‐SY5Y cells. We confirm that UV irradiation induces both apoptosis and necrosis in SH‐SY5Y cells and that phosphorylation of JNK at Thr183/Tyr185 in SH‐SY5Y cells treated with UV is an early event preceding apoptosis. We also demonstrate that phosphorylation of c‐Jun at Ser63 is an early event coinciding with JNK activation, and that the phosphorylation of c‐Jun is partially prevented by the JNK inhibitor SP600125. Despite the use of SP600125, the amount of cyt c released into the cytoplasm is not diminished and SP600125 is also unable to decrease the extent of UV‐induced apoptosis. These data support the hypothesis that in this system, UV‐induced apoptosis is not dependent exclusively on JNK activation. Possible involvement of cyclin‐dependent kinases (CDKs) in c‐Jun phosphorylation at Ser63 was excluded by pretreating UV‐irradiated SH‐SY5Y cells with the CDK1/2/5 inhibitor roscovitine.

Combining an amyloid‐beta (Aβ) cleaving enzyme inhibitor with a γ‐secretase modulator results in an additive reduction of Aβ production

A major hallmark of Alzheimers disease (AD) is the deposition of amyloid‐β (Aβ) peptides in amyloid plaques. Aβ peptides are produced by sequential cleavage of the amyloid precursor protein by the β amyloid cleaving enzyme (BACE) and the γ‐secretase (γ‐sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aβ42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ‐sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on‐ or off‐target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aβ42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aβ42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aβ production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.

Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

BackgroundDeregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimers disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems.MethodsHEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates.ResultsWestern blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line.ConclusionA novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.

In vivo reduction of beta-amyloid peptides in the brain of Tg2576 mice after oral administration of a novel BACE1 inhibitor

daily for 7 days, then 16 mg daily for 7 days, followed by a co-administration period of 10 days with 125 mg avagacestat.Results: 32 and 34 subjects were randomized in the donepezil and galantamine study, respectively. Coadministration of avagacestat and donepezil or galantamine was associated with an increase in the geometric mean Cmax (14-21%) and AUC(TAU) (24-26%) of donepezil and/or galantamine; an increase not likely to be clinically relevant. One serious adverse event (SAE) was reported in healthy subjects co-administered avagacestat and galantamine for 10 days. On Day 25, a subject was found to have elevated lipase (5x ULN) and amylase (2x ULN) and was diagnosed with pancreatitis. The subject, who also had multiple gall stones, was admitted to the hospital, was treated, and recovered fully. Co-administration of avagacestat with either donepezil or galantamine in healthy subjects for short periods of timewas associated with a higher frequency of AEs than administration of donepezil or galantamine alone. The most common AEs in either study included headache, nausea, and epistaxis. Most AEs were mild in nature and all, except nasal congestion, resolved prior to study discharge. Conclusions: Co-administration of multiple 125 mg doses of avagacestat (the highest dose tested in phase IIb studies) and either donepezil or galantamine at steady-state appeared to be generally well tolerated in healthy subjects. Co-administration of avagacestat was not associated with clinically relevant increases in Cmax or AUC for either donepezil or galantamine.