Holger Patzke

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice

BackgroundA hallmark of Alzheimer’s disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aβ42 and Aβ40. Many drug discovery efforts have focused on decreasing the production of Aβ42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aβ production to favor shorter, less amyloidogenic peptides than Aβ42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer’s disease.ResultsHere we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aβ42 in H4 cells (IC50 = 67 nM) and increased the shorter Aβ38 by 1.7 fold at the IC50 for lowering of Aβ42. AβTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aβ42 and did not alter AβTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aβ deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aβ aggregates, amyloid plaques, inflammatory markers, and cognitive deficits.ConclusionsEVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aβ42, attenuated memory deficits, and reduced Aβ plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer’s disease.

Cdk5 sinks into ALS

Recent research points to an involvement of deregulated cdk5 activity in the pathogenesis of mutant SOD1-mediated disease. In addition, inhibition of this activity might promote motor neuron survival. These observations have opened the door to further research into the role of cdk5 in ALS and other neurodegenerative diseases.

Activity-Dependent p25 Generation Regulates Synaptic Plasticity and Aβ-Induced Cognitive Impairment

Cyclin-dependent kinase 5 regulates numerous neuronal functions with its activator, p35. Under neurotoxic conditions, p35 undergoes proteolytic cleavage to liberate p25, which has been implicated in various neurodegenerative diseases. Here, we show that p25 is generated following neuronal activity under physiological conditions in a GluN2B- and CaMKIIα-dependent manner. Moreover, we developed a knockin mouse model in which endogenous p35 is replaced with a calpain-resistant mutant p35 (Δp35KI) to prevent p25 generation. The Δp35KI mice exhibit impaired long-term depression and defective memory extinction, likely mediated through persistent GluA1 phosphorylation at Ser845. Finally, crossing the Δp35KI mice with the 5XFAD mouse model of Alzheimers disease (AD) resulted in an amelioration of β-amyloid (Aβ)-induced synaptic depression and cognitive impairment. Together, these results reveal a physiological role of p25 production in synaptic plasticity and memory and provide new insights into the function of p25 in Aβ-associated neurotoxicity and AD-like pathology.

Partial Rescue of the p35−/− Brain Phenotype by Low Expression of a Neuronal-Specific Enolase p25 Transgene

Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca2+-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimers disease and may contribute to A-β peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35−/− phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.

Experience Modulates the Effects of Histone Deacetylase Inhibitors on Gene and Protein Expression in the Hippocampus: Impaired Plasticity in Aging

The therapeutic potential of histone deacetylase inhibitor (HDACi) treatment has attracted considerable attention in the emerging area of cognitive neuroepigenetics. The possibility that ongoing cognitive experience importantly regulates the cell biological effects of HDACi administration, however, has not been systematically examined. In an initial experiment addressing this issue, we tested whether water maze training influences the gene expression response to acute systemic HDACi administration in the young adult rat hippocampus. Training powerfully modulated the response to HDACi treatment, increasing the total number of genes regulated to nearly 3000, including many not typically linked to neural plasticity, compared with <300 following HDACi administration alone. Although water maze training itself also regulated nearly 1800 genes, the specific mRNAs, gene networks, and biological pathways involved were largely distinct when the same experience was provided together with HDACi administration. Next, we tested whether the synaptic protein response to HDACi treatment is similarly dependent on recent cognitive experience, and whether this plasticity is altered in aged rats with memory impairment. Whereas synaptic protein labeling in the young hippocampus was selectively increased when HDACi administration was provided in conjunction with water maze training, combined treatment had no effect on synaptic proteins in the aged hippocampus. Our findings indicate that ongoing experience potently regulates the molecular consequences of HDACi treatment and that the interaction of recent cognitive experience with histone acetylation dynamics is disrupted in the aged hippocampus. SIGNIFICANCE STATEMENT The possibility that interventions targeting epigenetic regulation could be effective in treating a range of neurodegenerative disorders has attracted considerable interest. Here we demonstrate in the rat hippocampus that ongoing experience powerfully modifies the molecular response to one such intervention, histone deacetylase inhibitor (HDACi) administration. A single learning episode dramatically shifts the gene expression profile induced by acute HDACi treatment, yielding a qualitatively distinct hippocampal transcriptome compared with the influence of behavioral training alone. The downstream synaptic protein response to HDACi administration is similarly experience-dependent, and we report that this plasticity is disrupted in the aged hippocampus. The findings highlight that accommodating the modulatory influence of ongoing experience represents a challenge for therapeutic development in the area of cognitive neuroepigenetics.

α7-nAChR agonist enhances neural plasticity in the hippocampus via a GABAergic circuit.

Agonists of the α7-nicotinic acetylcholine receptor (α7-nAChR) have entered clinical trials as procognitive agents for treating schizophrenia and Alzheimers disease. The most advanced compounds are orthosteric agonists, which occupy the ligand binding site. At the molecular level, agonist activation of α7-nAChR is reasonably well understood. However, the consequences of activating α7-nAChRs on neural circuits underlying cognition remain elusive. Here we report that an α7-nAChR agonist (FRM-17848) enhances long-term potentiation (LTP) in rat septo-hippocampal slices far below the cellular EC50 but at a concentration that coincides with multiple functional outcome measures as we reported in Stoiljkovic M, Leventhal L, Chen A, Chen T, Driscoll R, Flood D, Hodgdon H, Hurst R, Nagy D, Piser T, Tang C, Townsend M, Tu Z, Bertrand D, Koenig G, Hajós M. Biochem Pharmacol 97: 576-589, 2015. In this same concentration range, we observed a significant increase in spontaneous γ-aminobutyric acid (GABA) inhibitory postsynaptic currents and a moderate suppression of excitability in whole cell recordings from rat CA1 pyramidal neurons. This modulation of GABAergic activity is necessary for the LTP-enhancing effects of FRM-17848, since inhibiting GABAA α5-subunit-containing receptors fully reversed the effects of the α7-nAChR agonist. These data suggest that α7-nAChR agonists may increase synaptic plasticity in hippocampal slices, at least in part, through a circuit-level enhancement of a specific subtype of GABAergic receptor.

GSM treatment after the onset of Aβ deposition in mice reduces total Aβ load

Background:Alzheimer’s disease (AD) is a common, debilitating neurodegenerative disorder afflicting the aging population. Synaptic accumulation of Aß-protein oligomers and tau might be responsible for the neurological damage in AD. Thus, special interest has emerged in developing treatments that reduce the formation or facilitate the clearance of these oligomers. Utilizing molecular modeling and structure based design techniques we generated a series of novel organic heterocyclic compounds that are capable of recognizing the aggregated Aß-protein molecule and promoting clearance. Using cell-free and cell-based assays, a compound denominated NPT-400 with activity in the high nanomolar range was identified for pharmacokinetic (PK) and preliminary efficacy studies in APP transgenic (tg) mice. Methods: The mThy1-hAPP751 transgenic mouse was utilized for efficacy studies. This mouse combines the Swedish (K670M/N671L) and London (V717I) APPmutations, and these animals develop AD-like neuropathology and behavioral deficits starting at 3-4 months of age. Female non-tg and tg mice (w5 mo of age) received a single injection of vehicle or NPT-400-3 (IP, 10 mg/kg) three times per week for 6.5 weeks. Behavioral assessments were conducted during week 4 of treatment including locomotor activity and water maze. At the completion of treatment, brains were hemisected and tissue sections were processed for immunolabeling and confocal analysis, and homogenates were analyzed for levels of Abeta-protein by ELISA and immunoblot. For PK studies, wildtype C57Bl6 mice received IVor PO NPT-400-3 compound at 10 mg/kg and blood and plasma levels were analyzed at 0-24 hrs. Results: Treatment with NPT-400-3 resulted in a statistically significant normalization of locomotor activity accompanied by amelioration of the synaptic and dendritic pathology in the neocortex and hippocampus. By immunoblot and ELISA the levels of monomeric and oligomeric Aß-protein were statistically significantly reduced in the brains of the APP tg mice. PK studies showed that NPT400-3 is stable in plasma, is orally bioavailable and penetrates the brain with a B/P ratio of 0.8. Conclusions:NPT-400-3 is an orally bioavailable, brain penetrating Aß-protein stabilizing organic compound that reduces the accumulation of Aß-protein oligomers and ameliorates behavioral deficits and neuropathology in APP tg mice without overt adverse effects.

Pharmacokinetic and pharmacodynamic analysis of the gamma-secretase modulator (GSM) EVP-0015962

Background: CAD106 is an active immunotherapy developed for Alzheimer’s disease. It comprises amino acids 1-6 of Ab coupled to a virus-like particle derived from E. coli bacteriophage Qb. In APP overexpressing mice, administration of CAD106 induced Ab antibodies as well as a reduction of amyloid accumulation in the brain. Infusion of antibodies or active immunization against Ab have been shown to increase the level of total Ab in plasma indicating target engagement by the antibodies. In the present study we have investigated the active Ab immunotherapy CAD106 for its effect on plasma Ab. Methods: Cynomolgus monkeys aged 3 to 6 years were administered 600 mg CAD106 per subcutaneous or intramuscular injection at days 1, 14, 28 and every 4 weeks thereafter for a total of 24 weeks. Each dose contained either Alum or MF58 as adjuvant. Controls received adjuvant only. Total plasma Ab was determined at baseline and 12 to 14 days after the injections at week 8 and 24 using two different ELISAs and Western blotting. To minimize an interference of the CAD106-induced antibodies samples were SDSdenatured prior to quantification with an ELISA using a N-terminal capturing antibody. Alternatively, an assay combining C-terminal capture and mid-region detection antibodies was used. In addition, results were verified by direct SDS-PAGE separation of plasma followed by Western blotting. Results: Following active immunotherapy with CAD106, total plasma Ab levels increased compared to baseline and further between the intermediate and final time points of the study. Individual animals showed increases of up to about 50-fold over baseline. Median group increases reached up to 25-fold. Very similar results were obtained with the different assays. Overall the Ab levels in plasma correlated well with the Ab antibody titers. Conclusions: The increase in plasma Ab indicates an interaction of CAD106 induced antibodies with Ab in vivo. Its extent is in line with observations made with Nterminal monoclonal antibodies.

Design, Synthesis, and Evaluation of a Novel Series of Oxadiazine Gamma Secretase Modulators for Familial Alzheimer’s Disease

Herein we describe the design, synthesis, and evaluation of a novel series of oxadiazine-based gamma secretase modulators obtained via isosteric amide replacement and critical consideration of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent predicted CNS drug-like properties and desirable ADME/PK profile. This lead compound demonstrated robust Aβ42 reductions and subsequent Aβ37 increases in both rodent brain and CSF at 30 mg/kg dosed orally.

Generation and Partial Characterization of Rabbit Monoclonal Antibody to Amyloid-β Peptide 1–37 (Aβ37)

Secreted soluble amyloid-β 1-37 (Aβ37) peptide is one of the prominent Aβ forms next to Aβ40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aβ37 levels in combination with Aβ38, Aβ40, and Aβ42 to support the diagnosis of patients with probable Alzheimers disease (AD), and the value of antibody to Aβ37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aβ37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aβ37, and 2) to determine whether the antibody detects changes in Aβ37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aβ37 was found to be specific to Aβ37, since it did not react with Aβ36, Aβ38, Aβ39, Aβ40, and Aβ42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aβ37. The antibody was sensitive enough to measure CSF and plasma Aβ37 levels in ELISA. Immunohistological studies showed the presence of Aβ37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aβ37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.