Sue H. Lee

Amyloid-β Pathology and APOE Genotype Modulate Retinoid X Receptor Agonist Activity in Vivo

Background: Human APOE effects on RXR agonist activity are unclear. Results: In RXR agonist-treated EFAD mice, beneficial effects in APOE4 hippocampus include ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. Detrimental effects in APOE3 and APOE4 cortex might be from systemic hepatomegaly. Conclusion: Aβ pathology and APOE genotype modulate RXR agonist effects. Significance: Although promising for later stage Alzheimer disease, detrimental side effects limit translational application of RXR agonists. Previous data demonstrate that bexarotene (Bex), retinoid X receptor (RXR) agonist, reduces soluble and insoluble amyloid-β (Aβ) in Alzheimer disease (AD)-transgenic mice either by increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. However, although the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aβ, likely the proximal AD neurotoxin. If the APOE4 loss-of-function is lipidation of apoE4, then induction of ABCA1/ABCG1 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aβ42 with h-APOE), levels of soluble Aβ (Aβ42 and oligomeric Aβ) are highest in E4FAD hippocampus (HP) > E3FAD-HP > E4FAD cortex (CX) > E3FAD-CX, whereas levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6 months). In E4FAD-HP, short-term RXR agonist treatment (Bex or LG100268; 5.75–6 months) increased ABCA1, apoE4 lipoprotein-association/lipidation, and apoE4/Aβ complex, decreased soluble Aβ, and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and LG100268. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5–6 months) and actually increased soluble Aβ levels in E3FAD-CX and E4FAD-CX with the short-term protocol, possibly the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aβ pathology, i.e. low levels of apoE4 lipoprotein association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.

Targeting NO/cGMP Signaling in the CNS for Neurodegeneration and Alzheimer’s Disease

cAMP-response element-binding protein (CREB) plays a central role in various aspects of central nervous system (CNS) function, ranging from the developmental stages to neuronal plasticity and survival in adult brain. Activation of CREB plays a crucial role in learning and memory and is at the convergence of multiple intracellular signaling cascades including CAMKII and MAPK. This review focuses on the important functions of nitric oxide (NO) in activating CREB via the NO receptor, soluble guanylyl cyclase (sGC), and production of the second messenger, cGMP. The involvement of the NO/cGMP signaling pathway in synaptic plasticity suggests several avenues for therapeutic intervention, and targeting early synaptic degeneration could be an attractive approach for the development of novel disease-modifying approaches to treat cognition and memory dysfunction in neurodegenerative diseases.

A multifunctional therapeutic approach to disease modification in multiple familial mouse models and a novel sporadic model of Alzheimer’s disease

BackgroundClinical failures singularly targeting amyloid-β pathology indicate a critical need for alternative Alzheimer’s disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD.ResultsNMZ was tested in three mouse models of familial AD and one model of sporadic AD. In 3 × Tg hippocampal slices, NMZ restored LTP. In vivo, memory was improved with NMZ in all animal models with robust cognitive deficits. NMZ treatment lowered neurotoxic forms of Aβ in both APP/PS1 and 3 × Tg transgenic mice while also restoring neuronal plasticity biomarkers in the 3 × Tg mice. In EFAD mice, incorporation of the major genetic AD risk factor, hAPOE4, did not mute the beneficial drug effects. In a novel sporadic mouse model that manifests AD-like pathology caused by accelerated oxidative stress in the absence of any familial AD mutation, oral administration of NMZ attenuated hallmark AD pathology and restored biomarkers of synaptic and neuronal function.ConclusionsThe multifunctional approach, embodied by NMZ, was successful in mouse models of AD incorporating Aβ pathology (APP/PS1), tau pathology (3xTg), and APOE4, the major human genetic risk factor for AD (EFAD). The efficacy observed in a novel model of sporadic AD (Aldh2−/−) demonstrates that the therapeutic approach is not limited to rare, familial AD genetic mutations. The multifunctional drug, NMZ, was not designed directly to target Aβ and tau pathology; however, the attenuation of this hallmark pathology suggests the approach to be a highly promising, disease-modifying strategy for AD and mixed pathology dementia.

Soluble guanylyl cyclase is a critical regulator of migraine-associated pain:

Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite – factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.

CHEMOPROTEOMIC APPROACH TO CHARACTERIZING THE ROLE OF 4-HNE IN ACCELERATED COGNITIVE IMPAIRMENT

Background: Oxidative stress leads to the increased production of lipid peroxidation products (LPx) such as 4-hydroxynonenal (4HNE) which accumulate in the brain. 4-HNE is an electrophilic LPx capable of forming protein adducts at reactive cysteine residues. These protein adducts can act as toxic secondary messengers resulting in the dysregulation of signaling pathways involved in mitochondrial and neuronal function. Proteins modified by the most common lipid peroxidation product (LPx), 4-HNE, are investigated in neuronal cells and a mouse model of age-dependent dementia in order to elucidate novel signaling pathways linked to cognitive decline. Methods: Novel probes and chemoproteomic methods are used to visualized specifically the proteins modified by 4-HNE including analytical analysis via iTRAQ. In addition behavior is evaluated in this novel mouse model of age-dependent dementia.Results:Byutilizingnovel fluorescent probes, 4-HNEmodificationwas visualized dose dependently in neuronal cells. In addition, an increase of 4-HNE-adductswas seen early in the development of dementia in conjunction with accelerated cognitive deficits as early as 3 months. A follow up of several proteins linked with dementia such as PIN1are being further investigated in order to understand and elucidate novel signaling pathways linked to cognitive decline. Conclusions: In this mouse model where early 4-HNE modification is seen in parallel with early subtle cognition deficits becomes a useful tool for identifying early biomarkers dysregulated with cognitive deficits despite the appearance of traditional pathology.

Pharmacological manipulation of cGMP and NO/cGMP in CNS drug discovery

The development of small molecule modulators of NO/cGMP signaling for use in the CNS has lagged far behind the use of such clinical agents in the periphery, despite the central role played by NO/cGMP in learning and memory, and the substantial evidence that this signaling pathway is perturbed in neurodegenerative disorders, including Alzheimers disease. The NO-chimeras, NMZ and Nitrosynapsin, have yielded beneficial and disease-modifying responses in multiple preclinical animal models, acting on GABAA and NMDA receptors, respectively, providing additional mechanisms of action relevant to synaptic and neuronal dysfunction. Several inhibitors of cGMP-specific phosphodiesterases (PDE) have replicated some of the actions of these NO-chimeras in the CNS. There is no evidence that nitrate tolerance is a phenomenon relevant to the CNS actions of NO-chimeras, and studies on nitroglycerin in the periphery continue to challenge the dogma of nitrate tolerance mechanisms. Hybrid nitrates have shown much promise in the periphery and CNS, but to date only one treatment has received FDA approval, for glaucoma. The potential for allosteric modulation of soluble guanylate cyclase (sGC) in brain disorders has not yet been fully explored nor exploited; whereas multiple applications of PDE inhibitors have been explored and many have stalled in clinical trials.

SELECTIVE CALPAIN-1 VERSUS CALPAIN-1/CATHEPSIN-B DUAL INHIBITION AS A THERAPEUTIC APPROACH TO AD

C-terminal end of Ng. The present work was aimed at identifying enzymes generating this cleavage pattern. Methods:Two quenched fluorogenic peptides based on sequences of the main cleavage regions were used to test enzymes known to be upregulated in AD as well as probe fractionated mouse brain extracts for Ng-cleaving activity. The Ng fragments from in vitro cleavage were determined by MALDI-TOF MS and LC-ESI-MS. Results:We identified calpain I as cleaving Ng in its central region, at the C-terminal end of amino acids 37, 42 and 65. Fragments starting at amino acid 43 and smaller were among the dominant fragments seen by IPMS in CSF.We also identified the intracellular enzyme prolyl endopeptidase as being able to generate fragments lacking the very Cterminal three amino acids, which are also seen in CSF. While shorter Ng fragments were readily cleaved in vitro by prolyl endopeptidase, the efficiency of cleavage on larger Ng fragments was much lower. Conclusions: Calpain I and prolyl endopeptidase were identified as candidate enzymes involved in the formation of endogenous Ng peptides found in CSF, cleaving in the central region near the IQ domain and after Ng75. Whereas full length Ng is able to bind to calmodulin in the absence of Ca ions, most of the calpain fragments of Ng in CSF are lacking the IQ domain and are thus unable to do so. While the action of prolyl endopeptidase can explain the appearance of fragments lacking the last three C-terminal amino acids, the functional significance of that activity is still unclear. However, calpainand prolyl endopeptidase-specific fragments of Ng may give clues to increased activities of these enzymes during progression of AD.

OPTIMIZATION OF TISSUE-SELECTIVE ABCA1 AGONISTS (TSAAGS) AS AN EFFECTIVE AND TOLERATED THERAPY FOR ALZHEIMER’S DISEASE

Background:Alzheimer’s disease and related dementia (ADRD) is a multifactorial disease presenting a challenge to drug discovery. The greatest known risk factor is the APOE4-allele with 50% and 90% risk factors for heterozygotes and homozygotes, respectively. In the brain, ABCA1 initiates the assembly of apolipoprotein E (ApoE) containing lipoprotein-particles that transport lipids throughout the CNS. ApoE4 particles are less stable and poorly lipidated, contributing to loss of function in AD, which can be mitigated by ABCA1 overexpression. One amyloid-b (Ab) related mechanism relevant to APOE4 carriers is reduced clearance of Ab. Whereas therapeutic approaches solely targeting amyloid-b (Ab) have failed in the clinic, there remains a lack of therapeutic strategies incorporating the APOE4 risk factor. We propose to restore ApoE function by promoting its lipidation using an effective and well-tolerated tissue-selective ABCA1 agonist (TSAAg), addressing the multifactorial nature of ADRD, and effective in APOE4 carriers. Methods: Previous approaches to enhance apoE function using RXR/LXR agonists, ameliorated AD-related pathology in some mouse models, but induced detrimental lipogenic activation. Therefore, we have taken an innovative functional approach to restore the function of ApoE by screening for TSAAgs, that elevate ABCA1 in astrocytes and counter-screen against SREBP1c activation in hepatocytes to mitigate against detrimental lipogenic gene activation. Screening conditions have been optimized for Z’-factors > 0.8. Selected hits with neutral lipogenic actions were further profiled based upon multifactorial properties for cholesterol metabolism; anti-inflammatory, and insulin-sensitizing effects. Toxicodynamic and pharmacodynamics (TD/PD) studies were performed in WT, high-fat-diet-fed mice and in an AD-model. Results: From initial 25,000-compounds, 5 Chemotypes were selected for SAR and probed in primary astrocytes for regulation of cholesterol efflux and genes associated with neuroinflammation, energy utilization, insulin sensitivity, and cholesterol metabolism. Using liver, plasma, and brain biomarkers, TD/PD data and target-engagement were validated in vivo, compatible with in vitro data. Remarkably, in both mouse models, oral administration of TSAAg reversed cognitive deficits. Conclusions:A Preclinical Proof-Of-Concept was established. We propose that this functional approach has potential to impact multiple factors that contribute to ADRD, both associated with and not directly associated with Ab, in the highly vulnerable APOE4 population.

Activation of Nrf2 and Hypoxic Adaptive Response Contribute to Neuroprotection Elicited by Phenylhydroxamic Acid Selective HDAC6 Inhibitors

Activation of HIF-1α and Nrf2 is a primary component of cellular response to oxidative stress, and activation of HIF-1α and Nrf2 provides neuroprotection in models of neurodegenerative disorders, including ischemic stroke, Alzheimers and Parkinsons diseases. Screening a library of CNS-targeted drugs using novel reporters for HIF-1α and Nrf2 elevation in neuronal cells revealed histone deacetylase (HDAC) inhibitors as potential activators of these pathways. We report the identification of phenylhydroxamates as single agents exhibiting tripartite inhibition of HDAC6, inhibition of HIF-1 prolyl hydroxylase (PHD), and activation of Nrf2. Two superior tripartite agents, ING-6 and ING-66, showed neuroprotection against various cellular insults, associated with stabilization of both Nrf2 and HIF-1, and expression of their respective target genes in vitro and in vivo. Discovery of the innate ability of phenylhydroxamate HDAC inhibitors to activate Nrf2 and HIF provides a novel route to multifunctional neuroprotective agents and cautions against HDAC6 selective inhibitors as chemical probes of specific HDAC isoform function.

Erratum: A multifunctional therapeutic approach to disease modification in multiple familial mouse models and a novel sporadic model of Alzheimer's disease (Molecular Neurodegeneration (2016) 11 (35) DOI: 10.1186/s13024-016-0103-6)

Author details Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA. Department of Psychiatry, Neuropsychiatric Institute, University of Illinois at Chicago, Chicago, IL, USA. Department of Pathology, The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, NY, USA. Department of Biomedical & Molecular Sciences, Faculty of Health Sciences, Queen’s University, Kingston, ON, Canada. Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA.