iPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype.

BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.

Three-dimensional bioprinting of stem cell-derived central nervous system cells enables astrocyte growth, vasculogenesis, and enhances neural differentiation/function.

Current research tools for preclinical drug development such as rodent models and two-dimensional immortalized monocultures have failed to serve as effective translational models for human central nervous system (CNS) disorders. Recent advancements in the development of induced pluripotent stem cells (iPSCs) and three-dimensional (3D) culturing can improve the in vivo-relevance of preclinical models, while generating 3D cultures though novel bioprinting technologies can offer increased scalability and replicability. As such, there is a need to develop platforms that combine iPSC-derived cells with 3D bioprinting to produce scalable, tunable, and biomimetic cultures for preclinical drug discovery applications. We report a biocompatible poly(ethylene glycol)-based matrix which incorporates Arg-Gly-Asp and Tyr-Ile-Gly-Ser-Arg peptide motifs and full-length collagen IV at a stiffness similar to the human brain (1.5 kPa). Using a high-throughput commercial bioprinter we report the viable culture and morphological development of monocultured iPSC-derived astrocytes, brain microvascular endothelial-like cells, neural progenitors, and neurons in our novel matrix. We also show that this system supports endothelial-like vasculogenesis and enhances neural differentiation and spontaneous activity. This platform forms a foundation for more complex, multicellular models to facilitate high-throughput translational drug discovery for CNS disorders.

Understanding In Vitro Pathways to Drug Discovery for TDP-43 Proteinopathies.

The use of cellular models is a common means to investigate the potency of therapeutics in pre-clinical drug discovery. However, there is currently no consensus on which model most accurately replicates key aspects of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathology, such as accumulation of insoluble, cytoplasmic transactive response DNA-binding protein (TDP-43) and the formation of insoluble stress granules. Given this, we characterised two TDP-43 proteinopathy cellular models that were based on different aetiologies of disease. The first was a sodium arsenite-induced chronic oxidative stress model and the second expressed a disease-relevant TDP-43 mutation (TDP-43 M337V). The sodium arsenite model displayed most aspects of TDP-43, stress granule and ubiquitin pathology seen in human ALS/FTD donor tissue, whereas the mutant cell line only modelled some aspects. When these two cellular models were exposed to small molecule chemical probes, different effects were observed across the two models. For example, a previously disclosed sulfonamide compound decreased cytoplasmic TDP-43 and increased soluble levels of stress granule marker TIA-1 in the cellular stress model without impacting these levels in the mutant cell line. This study highlights the challenges of using cellular models in lead development during drug discovery for ALS and FTD and reinforces the need to perform assessments of novel therapeutics across a variety of cell lines and aetiological models.

Senolytics: A Novel Strategy for Neuroprotection in ALS?

Amyotrophic lateral sclerosis (ALS) is a progressive motor neurodegenerative disease that currently has no cure and has few effective treatments. On a cellular level, ALS manifests through significant changes in the proper function of astrocytes, microglia, motor neurons, and other central nervous system (CNS) cells, leading to excess neuroinflammation and neurodegeneration. Damage to the upper and lower motor neurons results in neural and muscular dysfunction, leading to death most often due to respiratory paralysis. A new therapeutic strategy is targeting glial cells affected by senescence, which contribute to motor neuron degeneration. Whilst this new therapeutic approach holds much promise, it is yet to be trialled in ALS-relevant preclinical models and needs to be designed carefully to ensure selectivity. This review summarizes the pathways involved in ALS-related senescence, as well as known senolytic agents and their mechanisms of action, all of which may inform strategies for ALS-focused drug discovery efforts.

Synthesis and in vitro evaluation of fluorine-18 benzimidazole sulfones as CB2 PET-radioligands.

Cannabinoid type 2 receptor (CB2) is up-regulated on activated microglial cells and can potentially be used as a biomarker for PET-imaging of neuroinflammation. In this study the synthesis and pharmacological evaluation of novel fluorinated pyridyl and ethyl sulfone analogues of 2-(tert-butyl)-5-((2-fluoropyridin-4-yl)sulfonyl)-1-(2-methylpentyl)-1H-benzo[d]imidazole (rac-1a) are described. In general, the ligands showed low nanomolar potency (CB2 EC50 < 10 nM) and excellent selectivity over the CB1 subtype (>10 000×). Selected ligands 1d, 1e, 1g and 3l showing high CB2 binding affinity (Ki < 10 nM) were radiolabelled with fluorine-18 from chloropyridyl and alkyl tosylate precursors with good to high isolated radioactive yields (25-44%, non-decay corrected, at the end of synthesis). CB2-specific binding of the radioligand candidates [18F]-1d and [18F]-3l was assessed on rat spleen cryosections using in vitro autoradiography. The results warrant further in vivo evaluation of the tracer candidates as prospective CB2 PET-imaging agents.

Recent Developments in TSPO PET Imaging as A Biomarker of Neuroinflammation in Neurodegenerative Disorders.

Neuroinflammation is an inflammatory response in the brain and spinal cord, which can involve the activation of microglia and astrocytes. It is a common feature of many central nervous system disorders, including a range of neurodegenerative disorders. An overlap between activated microglia, pro-inflammatory cytokines and translocator protein (TSPO) ligand binding was shown in early animal studies of neurodegeneration. These findings have been translated in clinical studies, where increases in TSPO positron emission tomography (PET) signal occur in disease-relevant areas across a broad spectrum of neurodegenerative diseases. While this supports the use of TSPO PET as a biomarker to monitor response in clinical trials of novel neurodegenerative therapeutics, the clinical utility of current TSPO PET radioligands has been hampered by the lack of high affinity binding to a prevalent form of polymorphic TSPO (A147T) compared to wild type TSPO. This review details recent developments in exploration of ligand-sensitivity to A147T TSPO that have yielded ligands with improved clinical utility. In addition to developing a non-discriminating TSPO ligand, the final frontier of TSPO biomarker research requires developing an understanding of the cellular and functional interpretation of the TSPO PET signal. Recent insights resulting from single cell analysis of microglial phenotypes are reviewed.

Pharmacological evaluation of a novel series of urea, thiourea and guanidine derivatives as P2X7 receptor antagonists.

We report on P2X7 receptor antagonists based on a lead adamantly-cyanoguanidine-aryl moiety. We have investigated the importance of the central cyanoguanidine moiety by replacing it with urea, thiourea or guanidine moieties. We have also investigated the linker length between the central moiety and the aryl portion. All compounds were assessed for their inhibitory potency in a pore-formation dye uptake assay at the P2X7 receptor. None of the compounds resulted in an improved potency illustrating the importance of the cyanoguanidine moiety in this chemotype.

TSPO as a target for glioblastoma therapeutics.

The translocator protein (TSPO) is an 18-kDa five-transmembrane protein, which is primarily found in the outer mitochondrial membrane. Levels of this protein are up-regulated in the most aggressive and common glioma, glioblastoma multiforme (GM). Levels of TSPO also correlate with GM clinical outcome, suggesting that TSPO may be a novel GM diagnostic imaging agent. Therapeutically, targeting the TSPO may provide a mechanism to abrogate the apoptotic-resistant, invasive and aggressive nature of GM and may also provide a way of targeting other anti-cancer treatments to GM sites. This review highlights recent progress in research on TSPO-based diagnostic imaging and therapeutics for GM.

An Investigation on Linker Modifications of Cyanoguanidine-Based P2X7 Receptor Antagonists.

Despite their acknowledged significance in the inflammatory signalling cascade across a range of disease states, P2X7R antagonists have not yet proven to be effective in clinical trials. In this study, we present findings on P2X7 receptor antagonists that are based on a core adamantyl-cyanoguanidine-quinoline lead. To investigate the specific features of the cyanoguanidine moiety that influence compound potency we carried out a structure-activity relationship (SAR) study. Compound potency was assessed using an in vitro dye-uptake assay measuring P2X7R pore formation. While none of the compounds displayed superior potency to the lead, we established key structural requirements for potent P2X7R antagonism. An additional SAR using different aryl groups was performed based on the promising activity displayed by the squaramide derivative.

Synthesis and Antitumour Evaluation of Tricyclic Indole-2-Carboxamides against Paediatric Brain Cancer Cells.

Antitumour properties of some cannabinoids (CB) have been reported in the literature as early as 1970s, however there is no clear consensus to date on the exact mechanisms leading to cancer cell death. The indole-based WIN 55,212-2 and SDB-001 are both known as potent agonists at both CB1 and CB2 receptors, yet we demonstrate herein that only the former can exert in vitro antitumour effects when tested against a paediatric brain cancer cell line KNS42. In this report, we describe the synthesis of novel 3,4-fused tricyclic indoles and evaluate their functional potencies at both cannabinoid receptors, as well as their abilities to inhibit the growth or proliferation of KNS42 cells. Compared to our previously reported indole-2-carboxamides, these 3,4-fused tricyclic indoles had either completely lost activities, or, showed moderate-to-weak antagonism at both CB1 and CB2 receptors. Compound 23 displayed the most potent antitumour properties among the series. Our results further support the involvement of non-CB pathways for the observed antitumour activities of amidoalkylindole-based cannabinoids, in line with our previous findings. Transcriptomic analysis comparing cells treated or non-treated with compound 23 suggested the observed antitumour effects of 23 are likely to result mainly from disruption of the FOXM1-regulated cell cycle pathways.

N-substituted 8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes as σ receptor ligands with potential neuroprotective effects.

Several libraries of similarly N-substituted 8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes (9), N-methyl-8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes (14), and N-methyl-11-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecan-8-ones (13) were synthesised and screened against a panel of CNS targets in order to develop structure-affinity relationships for cage-modified trishomocubane σ receptor ligands based on the N-substituted 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ol (8) scaffold. In general, compared to the corresponding 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ols, compounds of type 9 were potent σ receptor ligands with low levels of subtype selectivity, while the corresponding N-methyl-8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes showed reduced affinity but greater selectivity for σ2 receptors. The N-methyl-11-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecan-8-ones demonstrated the poorest σ receptor affinities, suggesting that 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ols interact with σ receptors in the bridged hemiaminal form rather than as the non-transannular, aminoketone tautomers. Several compounds of type 8, 9, and 14 were assessed for their ability to inhibit nitric oxide release in vitro, and demonstrated comparable or greater efficacy than 4-phenyl-1-(4-phenylbutyl)piperidine (PPBP), an established neuroprotective σ ligand with NOS inhibitory activity.

Synthesis and Preclinical Evaluation of Fluorinated 5-Azaindoles as CB2 PET Radioligands.

Several classes of cannabinoid receptor type 2 radioligands have been evaluated for imaging of neuroinflammation, with successful clinical translation yet to take place. Here we describe the synthesis of fluorinated 5-azaindoles and pharmacological characterization and in vivo evaluation of 18F-radiolabeled analogues. [18F]2 (hCB2 Ki = 96.5 nM) and [18F]9 (hCB2 Ki = 7.7 nM) were prepared using Cu-mediated 18F-fluorination with non-decay-corrected radiochemical yields of 15 ± 6% and 18 ± 2% over 85 and 80 min, respectively, with high radiochemical purities (>97%) and molar activities (140-416 GBq/µmol). In PET imaging studies in rats, both [18F]2 and [18F]9 demonstrated specific binding in CB2-rich spleen after pretreatment with CB2-specific GW405833. Moreover, [18F]9 exhibited higher brain uptake at later time points in a murine model of neuroinflammation compared with a healthy control group. The results suggest further evaluation of azaindole based CB2 radioligands is warranted in other neuroinflammation models.

Perphenazine-Macrocycle Conjugates Rapidly Sequester the Aß42 Monomer and Prevent Formation of Toxic Oligomers and Amyloid.

Alzheimer's disease is imposing a growing social and economic burden worldwide, and effective therapies are urgently required. One possible approach to modulation of the disease outcome is to use small molecules to limit the conversion of monomeric amyloid (Aß42) to cytotoxic amyloid oligomers and fibrils. We have synthesized modulators of amyloid assembly that are unlike others studied to date: these compounds act primarily by sequestering the Aß42 monomer. We provide kinetic and nuclear magnetic resonance data showing that these perphenazine conjugates divert the Aß42 monomer into amorphous aggregates that are not cytotoxic. Rapid monomer sequestration by the compounds reduces fibril assembly, even in the presence of pre-formed fibrillar seeds. The compounds are therefore also able to disrupt monomer-dependent secondary nucleation, the autocatalytic process that generates the majority of toxic oligomers. The inhibitors have a modular design that is easily varied, aiding future exploration and use of these tools to probe the impact of distinct Aß42 species populated during amyloid assembly.

Measuring Affinity of Ligands to the Oxytocin Receptor Using Radioligand Binding.

Two aims of oxytocin receptor (OTR)-targeted drug discovery are development of selective OTR-binding PET tracers and development of brain-permeable selective OTR agonists. By allowing measurement of central OTR binding site occupancy after administration of intranasal oxytocin, OTR PET tracers inform an understanding of the conflicting effects on pro-social behaviors seen with administration of intranasal oxytocin in human studies. By mitigating pharmacokinetic and pharmacodynamic limitations of intranasal oxytocin, development of brain-permeable selective OTR agonists may produce therapies for mental disorders that involve asocial symptoms. A key step in development of new OTR-targeting PET radioligands and small molecule agonists is measurement of OTR affinity. One technique that can quantitate the affinity of candidate ligands for the OTR is competition radioligand binding. This chapter describes the materials, methods, and considerations of experimental design required to conduct the steps of competition radioligand binding for OTR drug discovery.

Purinergic P2X7 Receptor: A Therapeutic Target in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by upper and lower motor neuron loss. The pathomechanisms of ALS are still poorly understood with current hypotheses involving genetic mutations, excitotoxicity, and reactive oxygen species formation. In the absence of a disease-altering clinically approved therapeutic, there is an ever-increasing need to identify new targets to develop drugs that delay disease onset and/or progression. The purinergic P2X7 receptor (P2X7R) has been implicated widely across the ALS realm, providing a potential therapeutic strategy. This review summarizes the current understanding of ALS, the P2X7R and its role in ALS, the current landscape of P2X7R antagonists, and the in vivo potential of these antagonists in preclinical ALS models.

Pharmacological characterization of a structural hybrid P2X7R antagonist using ATP and LL-37.

Antagonists of the P2X7 receptor (P2X7R) have the potential to treat diseases where neuroinflammation is present such as depression, chronic pain and Alzheimer's disease. We recently developed a structural hybrid (C1; 1-((adamantan-1-yl)methyl)-2-cyano-3-(quinolin-5-yl)guanidine) of a purported competitive P2X7R antagonist (C2; 2-cyano-1-((1S)-1-phenylethyl)-3-(quinolin-5-yl)guanidine) and a likely negative allosteric modulator (NAM) of the P2X7R (C3; N-((adamantan-1-yl)methyl)-2-chloro-5-methoxybenzamide). Here we aimed to pharmacologically characterize C1, to gain insights into how select structural components impact antagonist interaction with the P2X7R. A second aim was to examine the role of the peptide LL-37, an apparent activator of the P2X7R, and compare the ability of multiple P2X7R antagonists to block its effects. Compounds 1, 2 and 3 were characterised using washout, Schild and receptor protection studies, all using dye uptake assays in HEK293 cells expressing the P2X7R. LL-37 was examined in the same HEK293 cells and THP-1 monocytes. Compounds 2 and 3 acted as a BzATP-competitive antagonist and NAM of the P2X7R respectively. Compound 1 was a slowly reversible NAM of the P2X7R suggesting the incorporation of an appropriately positioned adamantane promotes binding to the allosteric site of the P2X7R. LL-37 was shown to potentiate the ability of ATP to induce dye uptake at low concentrations (1-3 µg mL-1) or induce dye uptake alone at higher concentrations (10-20 µg mL-1). None of the P2X7R antagonists studied were able to block LL-37-induced dye uptake bringing in to question the ability of current P2X7R antagonists to inhibit the inflammatory action of LL-37 in vivo.

Synthesis and antitumour evaluation of indole-2-carboxamides against paediatric brain cancer cells.

Paediatric glioblastomas are rapidly growing, devastating brain neoplasms with an invasive phenotype. Radiotherapy and chemotherapy, which are the current therapeutic adjuvant to surgical resection, are still associated with various toxicity profiles and only marginally improve the course of the disease and life expectancy. A considerable body of evidence supports the antitumour and apoptotic effects of certain cannabinoids, such as WIN55,212-2, against a wide spectrum of cancer cells, including gliomas. In fact, we previously highlighted the potent cytotoxic activity of the cannabinoid ligand 5 against glioblastoma KNS42 cells. Taken together, in this study, we designed, synthesised, and evaluated several indoles and indole bioisosteres for their antitumour activities. Compounds 8a, 8c, 8f, 12c, and 24d demonstrated significant inhibitory activities against the viability (IC50 = 2.34-9.06 µM) and proliferation (IC50 = 2.88-9.85 µM) of paediatric glioblastoma KNS42 cells. All five compounds further retained their antitumour activities against two atypical teratoid/rhabdoid tumour (AT/RT) cell lines. When tested against a medulloblastoma DAOY cell line, only 8c, 8f, 12c, and 24d maintained their viability inhibitory activities. The viability assay against non-neoplastic human fibroblast HFF1 cells suggested that compounds 8a, 8c, 8f, and 12c act selectively towards the panel of paediatric brain tumour cells. In contrast, compound 24d and WIN55,212-2 were highly toxic toward HFF1 cells. Due to their structural resemblance to known cannabimimetics, the most potent compounds were tested in cannabinoid 1 and 2 receptor (CB1R and CB2R) functional assays. Compounds 8a, 8c, and 12c failed to activate or antagonise both CB1R and CB2R, whereas compounds 8f and 24d antagonised CB1R and CB2R, respectively. We also performed a transcriptional analysis on KNS42 cells treated with our prototype compound 8a and highlighted a set of seven genes that were significantly downregulated. The expression levels of these genes were previously shown to be positively correlated with tumour growth and progression, indicating their implication in the antitumour activity of 8a. Overall, the drug-like and selective antitumour profiles of indole-2-carboxamides 8a, 8c, 8f, and 12c substantiate the versatility of the indole scaffold in cancer drug discovery.

Effect of age on proliferation-regulating factors in human adult neurogenic regions.

Neurogenesis, the birth of new neurons, continues throughout adulthood in the human subventricular zone (SVZ) and hippocampus. It is not known how levels of putative proliferation-regulating factors change with age in human adult neurogenic areas. The current project employed ELISAs to investigate changes in levels of putative proliferation-regulating factors in the healthy human SVZ and dentate gyrus throughout the adult lifespan (18-104 years). Levels of brain-derived neurotrophic factor, basic fibroblast growth factor and interleukin (IL)-1ß were significantly higher in the hippocampus than in the SVZ and levels of glial-derived neurotrophic factor and transforming growth factor-α were significantly higher in the SVZ (p < 0.005), suggesting that factors with predominant influences on neurogenesis differ between the two human adult neurogenic areas. Hippocampal levels of transforming growth factor-ß1 strongly increased with age (n = 9, p < 0.01), whereas hippocampal and SVZ levels of brain-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, glial-derived neurotrophic factor, heparin-binding epidermal growth factor, insulin-like growth factor-1, IL-1ß, IL-6 and transforming growth factor-α did not change significantly with age in the SVZ or hippocampus. These findings suggest regulation of the adult neurogenic environment in the human brain may differ over time from that in other species.

Reversing binding sensitivity to A147T translocator protein.

The translocator protein (TSPO) is a target for the development of neuroinflammation imaging agents. Clinical translation of TSPO PET ligands, such as [11C]DPA-713, has been hampered by the presence of a common polymorphism (A147T TSPO), at which all second-generation TSPO ligands lose affinity. Little is known about what drives binding at A147T compared to WT TSPO. This study aimed to identify moieties in DPA-713, and related derivatives, that influence binding at A147T compared to WT TSPO. We found changes to the nitrogen position and number in the heterocyclic core influences affinity to WT and A147T to a similar degree. Hydrogen bonding groups in molecules with an indole core improve binding at A147T compared to WT, a strategy that generated compounds that possess up to ten-times greater affinity for A147T. These results should inform the future design of compounds that bind both A147T and WT TSPO for use in neuroinflammation imaging.

Tricyclic heterocycles display diverse sensitivity to the A147T TSPO polymorphism.

The 18 kDa translocator protein (TSPO) is a target for the development of imaging agents to detect neuroinflammation. The clinical utility of second-generation TSPO ligands has been hindered by the presence of a polymorphism, rs6971, which causes a non-conservative substitution of alanine for threonine at amino acid residue 147 (TSPO A147T). Given the complex nature of TSPO binding, and the lack of non-discriminating high-affinity ligands at both wild type and A147T forms of TSPO, a series of novel TSPO ligands containing various heterocyclic scaffolds was developed to explore the pharmacophoric drivers of affinity loss at TSPO A147T. In general, N-benzyl-N-methyl-substituted amide ligands showed increased affinity at TSPO A147T, and a pyrazolopyrimidine acetamide containing this motif displayed low nanomolar binding affinities to both TSPO forms.