Three dimensional and four dimensional live imaging to study mechanisms of progressive neurodegeneration.

Neurodegenerative diseases are complex and progressive, posing challenges to their study and understanding. Recent advances in microscopy imaging technologies have enabled the exploration of neurons in three spatial dimensions (3D) over time (4D). When applied to 3D cultures, tissues, or animals, these technologies can provide valuable insights into the dynamic and spatial nature of neurodegenerative diseases.

Huntington's disease iPSC models-using human patient cells to understand the pathology caused by expanded CAG repeats.

A major advance in the study of Huntington's disease (HD) has been the development of human disease models employing induced pluripotent stem cells (iPSCs) derived from patients with HD. Because iPSCs provide an unlimited source of cells and can be obtained from large numbers of HD patients, they are a uniquely valuable tool for investigating disease mechanisms and for discovering potential disease-modifying therapeutics. Here, we summarize some of the important findings in HD pathophysiology that have emerged from studies of patient-derived iPSC lines.

Huntington's disease mouse models: unraveling the pathology caused by CAG repeat expansion.

Huntington's disease (HD) is a neurodegenerative disease that results in motor and cognitive dysfunction, leading to early death. HD is caused by an expansion of CAG repeats in the huntingtin gene (). Here, we review the mouse models of HD.

Approaches to develop therapeutics to treat frontotemporal dementia.

Frontotemporal degeneration (FTD) is a complex disease presenting as a spectrum of clinical disorders with progressive degeneration of frontal and temporal brain cortices and extensive neuroinflammation that result in personality and behavior changes, and eventually, death. There are currently no effective therapies for FTD. While 60-70% of FTD patients are sporadic cases, the other 30-40% are heritable (familial) cases linked to mutations in several known genes.

Cell death assays for neurodegenerative disease drug discovery.

: Neurodegenerative diseases affect millions of people worldwide. Neurodegeneration is gradual over time, characterized by neuronal death that causes deterioration of cognitive or motor functions, ultimately leading to the patient's death. Currently, there are no treatments that effectively slow the progression of any neurodegenerative disease, but improved microscopy assays and models for neurodegeneration could lead the way to the discovery of disease-modifying therapeutics.

Human iPS Cell-Derived Patient Tissues and 3D Cell Culture Part 2: Spheroids, Organoids, and Disease Modeling.

Human induced pluripotent stem cells (HiPSCs) provide several advantages for drug discovery, but principally they provide a source of clinically relevant tissue. Furthermore, the use of HiPSCs cultured in three-dimensional (3D) systems, as opposed to traditional two-dimensional (2D) culture approaches, better represents the complex tissue architecture in vivo. The use of HiPSCs in 3D spheroid and organoid culture is now growing, but particularly when using myocardial, intestinal enteric nervous system, and retinal cell lines.

Human iPS Cell-Derived Patient Tissues and 3D Cell Culture Part 1: Target Identification and Lead Optimization.

Human-induced pluripotent stem cells (HiPSCs), and new technologies to culture them into functional cell types and tissues, are now aiding drug discovery. Patient-derived HiPSCs can provide disease models that are more clinically relevant and so more predictive than the currently available animal-derived or tumor cell-derived cells. These cells, consequently, exhibit disease phenotypes close to the human pathology, particularly when cultured under conditions that allow them to recapitulate the tissue architecture in three-dimensional (3D) systems.

Screening for compounds that modulate epigenetic regulation of the transcriptome: an overview.

Epigenetic control of the transciptome is a complex and highly coordinated cellular process. One critical mechanism involves DNA methylation, mediated by distinct but related DNA methyltransferases (DNMTs). Although several DNMT inhibitors are available, most are nonselective; selective DNMT inhibitors, therefore, could be optimal as therapeutics, as well acting as chemical probes to elucidate the fundamental biology of individual DNMTs.

Drug discovery and the human kinome: recent trends.

A major new trend in drugs targeted at protein kinases is the discovery of allosteric modulators. These compounds differ from ATP-centric drugs in that they do not compete with ATP for binding to the catalytic domain, generally acting by inducing conformational changes to modulate activity. They could provide a number of advantages over more classical protein kinase drugs.

Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening.

Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest.

Human kinome drug discovery and the emerging importance of atypical allosteric inhibitors.

Importance Of The Field: Protein kinases are important targets for drug discovery because they possess critical roles in many human diseases. Several protein kinase inhibitors have entered clinical development with others having already been approved for treating a host of diseases. However, many kinase inhibitors suffer from non-selectivity because they interact with the ATP binding region which has similar structures amongst the protein kinases and this non-selectivity sometimes can cause side effects.

New insights into GPCR function: implications for HTS.

G protein-coupled receptors (GPCRs) are a large family of proteins that represent targets for approximately 40% of all approved drugs. They possess unique structural motifs that allow them to interact with a diverse series of extracellular ligands, as well as intracellular signaling proteins, such as G proteins, RAMPs, arrestins, and indeed other receptors. Extensive efforts are under way to discover new generations of drugs against GPCRs with unique targeted therapeutic uses, including "designer" drugs such as allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes.

The current status of drug discovery against the human kinome.

Protein kinases are important targets in drug discovery programs aimed at treating many devastating diseases, including cancer, autoimmune disorders, diabetes, and neurological disorders. Most "classical" drug discovery efforts employ rational drug design methods based upon structural information to identify compounds targeting the enzyme catalytic domain. Novel information on kinase biology is opening up other approaches in the design of selective inhibitors that may provide more subtle modulation of these drug discovery targets.

Photoproteins: important new tools in drug discovery.

The G protein-coupled receptor (GPCR) family is a major target for drug discovery, and most, if not all, GPCRs can couple to Ca2+ signaling. Consequently, there are a number of cellbased, primary, high-throughput screening (HTS) assays used for drug discovery that assess changes in intracellular Ca2+ as a functional readout of GPCR activation. Historically, changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to changes in intracellular Ca2+ concentration.

An overview of drug screening using primary and embryonic stem cells.

Cellular technologies are widely used in drug discovery to treat human diseases. Most studies involve the expression of recombinant targets in immortalized cells and measure drug interactions using simple, quantifiable responses. Such cells are also amenable to high throughput screening (HTS) methods.

The use of AlphaScreen technology in HTS: current status.

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx.

Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening.

Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses.

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease.

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells.

Cysteine proteases are the major beta-secretase in the regulated secretory pathway that provides most of the beta-amyloid in Alzheimer's disease: role of BACE 1 in the constitutive secretory pathway.

This article focuses on beta-amyloid (Abeta) peptide production and secretion in the regulated secretory pathway and how this process relates to accumulation of toxic Abeta in Alzheimer's disease. New findings are presented demonstrating that most of the Abeta is produced and secreted, in an activity-dependent manner, through the regulated secretory pathway in neurons. Only a minor portion of cellular Abeta is secreted via the basal, constitutive secretory pathway.

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography.

Endomorphin-1: induction of motor behavior and lack of receptor desensitization.

The endomorphins are recently discovered endogenous agonists for the mu-opioid receptor (Zadina et al., 1997). Endomorphins produce analgesia; however, their role in other brain functions has not been elucidated.

Template-constrained cyclic peptide analogues of somatostatin: subtype-selective binding to somatostatin receptors and antiangiogenic activity.

Beta-turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting biological activities, contains a beta-turn in its bioactive conformation. The beta-turn and biological activities of somatostatin have been succesfully mimicked in cyclic hexapeptide analogues.

Differential regulation of the cloned kappa and mu opioid receptors.

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response.

Fentanyl and its analogs desensitize the cloned mu opioid receptor.

Fentanyl, and its structural analogs lofentanil and sufentanil, are potent analgesics used clinically in the management of pain. However, the high analgesic potency of these compounds is limited by the development of tolerance after chronic use. To investigate whether their tolerance development may be related to mu receptor desensitization, the cloned mouse mu receptor as well as mutant forms of the receptor were stably expressed in HEK 293 cells and tested for their response to continuous opioid treatment.