Alzheimer's disease-related amyloid-β induces synaptotoxicity in human iPS cell-derived neurons

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer''s disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.In Alzheimer''s disease (AD), synapse damage and synapse loss are thought to underlie cognitive deficits.¹ Oligomers of the amyloid-β (Aβ) peptide appear to induce synaptic failure as an early event in the etiology of AD.^{2, 3, 4} However, despite its well-established synapse-impairing effects in rodent models,^{5, 6, 7} the synaptotoxic actions of Aβ most relevant for the human disease have not been identified in a human model system. Several studies have investigated the synaptotoxic effects of Aβ in cultured rodent neurons and in transgenic mouse models revealing a multitude of potential mechanisms affecting synapses. Postsynaptic Aβ actions result in the loss of functional (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type) glutamate receptors,^{8, 9, 10} involve long-term depression-like mechanisms,^{9, 11, 12} and lead to the degradation of the entire postsynapse (dendritic spines).^{9, 11, 13} In addition, several distinct presynaptic Aβ actions on the synaptic vesicle cycle have been described.^{10, 14} Furthermore, Aβ-induced impairments of axonal transport regulation and Aβ-induced axon degeneration have been found in rodent neurons.^{15, 16, 17} This puzzling diversity of Aβ-induced synapse-related defects raises the question whether all of them are involved in the early pathomechanisms of human AD.In addition to well-established animal systems, the modelling of human neurological disease pathologies by human induced pluripotent stem cell (hiPSC) technology¹⁸ has been proposed as an innovative approach.^{19, 20, 21} The in vitro differentiation of hiPSCs to excitable neurons has been reported using a variety of protocols.^{22, 23, 24} However, quantitative analysis of both functional glutamatergic and GABAergic synapses has been difficult to achieve.^{19, 25, 26} In addition to studying the functional properties of iPSC-derived human neurons from healthy individuals, the in vitro differentiation of patient-derived iPSCs has been used to model complex neurodevelopmental and neurodegenerative diseases.^{19, 27, 28} Recently, iPSCs derived from AD patients have been reported to exhibit increased secretion of Aβ upon in vitro neuronal differentiation; however, neither a loss of synapses nor an impairment of synapse function was detected.^{21, 29, 30, 31, 32, 33} Here we describe a hiPSC-based, carefully optimized in vitro differentiation protocol, including a novel immunopanning step, which enabled us to study the deleterious effects of application of Aβ on human cortical neurons and on human synapses.     

Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease

Chagas disease, caused by Trypanosoma cruzi, is an important global public health problem which, despite partial efficacy of benznidazole (Bz) in acute phase, urgently needs an effective treatment. Cardiotoxicity is a major safety concern for conduction of more accurate preclinical drug screening platforms. Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are a reliable model to study genetic and infectious cardiac alterations and may improve drug development. Herein, we introduce hiPSC-CM as a suitable model to study T. cruzi heart infection and to predict the safety and efficacy of anti-T. cruzi drugs.     

Amino-caprolactam γ-secretase inhibitors showing potential for the treatment of Alzheimer's disease

Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aβ_x-40 levels in FVB mice via a single 100 mpk IP dose are highlighted.     

Developing β-secretase inhibitors for treatment of Alzheimer's disease

β-Secretase (memapsin 2; BACE-1) is the first protease in the processing of amyloid precursor protein leading to the production of amyloid-β (Aβ) in the brain. It is believed that high levels of brain Aβ are responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, β-secretase is a major therapeutic target for the development of inhibitor drugs. During the past decade, steady progress has been made in the evolution of β-secretase inhibitors toward better drug properties. Recent inhibitors are potent, selective and have been shown to penetrate the blood-brain barrier to inhibit Aβ levels in the brains of experimental animals. Moreover, continuous administration of a β-secretase inhibitor was shown to rescue age-related cognitive decline in transgenic AD mice. A small number of β-secretase inhibitors have also entered early phase clinical trials. These developments offer some optimism for the clinical development of a disease-modifying drug for AD.     

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10h.     

Rapid and efficient screening of Alzheimer's disease β-amyloid inhibitors using label-free gold nanoparticles

Herein we report that a visual, label-free gold nanoparticle-based assay for rapid and efficient screening of Alzheimer's disease β-amyloid inhibitors.     

Structural basis of C-terminal β-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.     

Prevention of β-amyloid induced toxicity in human iPS cell-derived neurons by inhibition of Cyclin-dependent kinases and associated cell cycle events

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline due to the selective neuronal loss in the cortex and hippocampus of the brains. Generation of human induced pluoripotent stem (hiPS) cells holds great promise for disease modeling and drug discovery in AD. In this study, we used neurons with forebrain marker expression from two unrelated hiPS cell lines. As both populations of neurons were vulnerable to β-amyloid 1–42 (Aβ1–42) aggregates, a hallmark of AD pathology, we used them to investigate cellular mediators of Aβ1–42 toxicity. We observed in neurons differentiated from both hiPS cell lines that Aβ induced toxicity correlated with cell cycle re-entry and was inhibited by pharmacological inhibitors or shRNAs against Cyclin-dependent kinase 2 (Cdk2). As one of the hiPS cell lines has been developed commercially to supply large quantities of differentiated neurons (iCell® Neurons), we screened a chemical library containing several hundred compounds and discovered several small molecules as effective blockers against Aβ1–42 toxicity, including a Cdk2 inhibitor. To our knowledge, this is the first demonstration of an Aβ toxicity screen using hiPS cell-derived neurons. This study provided an excellent example of how hiPS cells can be used for disease modeling and high-throughput compound screening for neurodegenerative diseases.     

Anti-aβ therapeutics in Alzheimer's disease: the need for a paradigm shift

Most current Alzheimer's disease (AD) therapies in advanced phases of development target amyloid β-peptide (Aβ) production, aggregation, or accumulation. Translational models suggest that anti-Aβ therapies may be highly effective if tested as agents to prevent or delay development of the disease or as therapies for asymptomatic patients with very early signs of AD pathology. However, anti-Aβ therapeutics are currently being tested in symptomatic patients where they are likely to be much less effective or ineffective. The lack of alignment between human clinical studies and preclinical studies, together with predictions about optimal trial design based on our understanding of the initiating role of Aβ aggregates in AD, has created a treatment versus prevention dilemma. In this perspective, we discuss why it is imperative to resolve this dilemma and suggest ways for moving forward in the hopes of enhancing the development of truly effective AD therapeutics.     

Is immunotherapy an effective treatment for Alzheimer's disease?

Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. Current views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease. Animal models of the disease have provided positive results from both approaches. Thus, an initial clinical trial using immunization with human Aβ in AD patients was started, but then shortly halted because of an unusually high incidence (6%) of meningoencephalitis. A long and currently ongoing debate in the scientific community about the pro or contra of vaccination or passive immunization with Aβ in AD is thereafter started. Here, the authors would like to stress few points of concern regarding these approaches in clinical practice.     

Alzheimer's disease drug candidates stabilize A-β protein native structure by interacting with the hydrophobic core

Deposition of amyloid fibrils, consisting primarily of Aβ₄₀ and Aβ₄₂ peptides, in the extracellular space in the brain is a major characteristic of Alzheimer''s disease (AD). We recently developed new (to our knowledge) drug candidates for AD that inhibit the fibril formation of Aβ peptides and eliminate their neurotoxicity. We performed all-atom molecular-dynamics simulations on the Aβ₄₂ monomer at its α-helical conformation and a pentamer fibril fragment of Aβ₄₂ peptide with or without LRL and fluorene series compounds to investigate the mechanism of inhibition. The results show that the active drug candidates, LRL22 (EC₅₀ = 0.734 μM) and K162 (EC₅₀ = 0.080 μM), stabilize hydrophobic core I of Aβ₄₂ peptide (residues 17–21) to its α-helical conformation by interacting specifically in this region. The nonactive drug candidates, LRL27 (EC₅₀ > 10 μM) and K182 (EC₅₀ > 5 μM), have little to no similar effect. This explains the different behavior of the drug candidates in experiments. Of more importance, this phenomenon indicates that hydrophobic core I of the Aβ₄₂ peptide plays a major mechanistic role in the formation of amyloid fibrils, and paves the way for the development of new drugs against AD.     

TGFβ inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy

Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGFβ as a novel signal to regulate hematopoietic development, as the TGFβ inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.     

Structure guided P1' modifications of HEA derived β-secretase inhibitors for the treatment of Alzheimer's disease

The synthesis and SAR of a series of BACE-1 hydroxyethyl amine inhibitors containing substitutions on a spirocyclobutyl moiety is described. Selectivity against cathepsin D, a related aspartyl protease with potential off target toxicity, and improved microsomal stability is exemplified.     

Naproxen interferes with the assembly of Aβ oligomers implicated in Alzheimer's disease

Experimental and epidemiological studies have shown that the nonsteroidal antiinflammatory drug naproxen may be useful in the treatment of Alzheimer''s disease. To investigate the interactions of naproxen with Aβ dimers, which are the smallest cytotoxic aggregated Aβ peptide species, we use united atom implicit solvent model and exhaustive replica exchange molecular dynamics. We show that naproxen ligands bind to Aβ dimer and penetrate its volume interfering with the interpeptide interactions. As a result naproxen induces a destabilizing effect on Aβ dimer. By comparing the free-energy landscapes of naproxen interactions with Aβ dimers and fibrils, we conclude that this ligand has stronger antiaggregation potential against Aβ fibrils rather than against dimers. The analysis of naproxen binding energetics shows that the location of ligand binding sites in Aβ dimer is dictated by the Aβ amino acid sequence. Comparison of the in silico findings with experimental observations reveals potential limitations of naproxen as an effective therapeutic agent in the treatment of Alzheimer''s disease.     

In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease

This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.     

Immunoreactivity for β-endorphin in LH-RH neurons of the fetal human hypothalamus

Summary A peptide immunochemically related to -endorphin was detected in some LH-RH neurons of the fetal human hypothalamus by comparison of adjacent sections stained for -endorphin and for LH-RH. In the same section, by successive staining and after antibody elution, both peptides were again revealed in the same neuron. The significance of the presence of the -endorphin-like material in LH-RH neurons is discussed.     

Establishment of a pancreatic β cell proliferation model in vitro and a platform for diabetes drug screening

Diabetes, a disease resulting from loss of functional β cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating β cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic β cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic β cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic β cell proliferation model was further validated by a proliferation assay using differentiated pancreatic β cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic β cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic β cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.     

New approaches for the treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is the most prevalent chronic neurodegenerative disease. Current approved therapies are symptomatic treatments having some effect on cognitive function. Therapies that target β-amyloid (Aβ) have been the focus of efforts to develop a disease modification treatment for AD but these approaches have failed to show any clinical benefit so far. Beyond the ‘Aβ hypothesis’, there are a number of newer approaches to treat AD with neuroinflammation emerging as a very active area of research based on risk gene analysis. This short review will summarize approved drug therapies, recent clinical trials and new approaches for the treatment of AD.