Aβ-degrading proteases in the pathogenesis of late-onset Alzheimer disease

Alzheimer disease (AD) is a devastating neurodegenerative disorder, affecting almost 6 million people in the United States alone, characterized by abnormal accumulation of an aggregation-prone, variable-length peptide known as the amyloid-β protein (Aβ). Considerable progress has been made elucidating the molecular pathogenesis of rare forms of early-onset AD (EOAD) attributable to autosomal-dominant mutations, which in all cases have been found to affect the production of Aβ either by elevating the levels of all forms of Aβ or by increasing the relative proportion of longer, more amyloidogenic species (e.g., Aβ42) to shorter, less aggregation-prone species (e.g., Aβ40). Nevertheless, these familial forms of the disease account for <2% of all AD diagnoses, and the precise mechanistic cause(s) of the remaining >98% of late-onset AD (LOAD) cases remains poorly understood, apart from the identification of certain genetic and environmental factors affecting AD risk. This proposal explores the overall hypothesis that LOAD can be triggered by defective clearance of Aβ by one or more Aβ-degrading proteases (AβDPs). More specifically, we investigate the hypothesis that a transient increase in Aβ early in life can serve as a common pathogenic mechanism for LOAD risk factors. We demonstrate that a severe traumatic brain injury in novel model of LOAD, which lacks the genetic mutations associated with EOAD, can trigger an acute rise in Aβ as well as accumulation of Aβ fibrils by 1-day post-injury. Additionally, we identify cathepsin D (CatD), a lysosomal protease, as a novel AβDP, demonstrating that it is the most important in vivo regulator of intracellular, insoluble Aβ yet identified. Intriguingly, we discovered that CatD degrades Aβ42 and Aβ40 with markedly different kinetics, resulting in increases to the Aβ42/40 ratio resembling those produced by EOAD-linked mutations. Finally, we detail the successful development of high-throughput assays to measure the proteolytic degradation of different substrates for insulin-degrading enzyme (IDE). We use these assays, in combination with other methods, to discover novel peptidic and zinc-targeting inhibitors of IDE, which can serve as novel pharmacological tools to help elucidate the role of IDE in the pathogenesis of LOAD

Extracellular Vesicles in Type 1 Diabetes: A Versatile Tool

Type 1 diabetes is a chronic autoimmune disease affecting nearly 35 million people. This disease develops as T-cells continually attack the β-cells of the islets of Langerhans in the pancreas, which leads to β-cell death, and steadily decreasing secretion of insulin. Lowered levels of insulin minimize the uptake of glucose into cells, thus putting the body in a hyperglycemic state. Despite significant progress in the understanding of the pathophysiology of this disease, there is a need for novel developments in the diagnostics and management of type 1 diabetes. Extracellular vesicles (EVs) are lipid-bound nanoparticles that contain diverse content from their cell of origin and can be used as a biomarker for both the onset of diabetes and transplantation rejection. Furthermore, vesicles can be loaded with therapeutic cargo and delivered in conjunction with a transplant to increase cell survival and long-term outcomes. Crucially, several studies have linked EVs and their cargos to the progression of type 1 diabetes. As a result, gaining a better understanding of EVs would help researchers better comprehend the utility of EVs in regulating and understanding type 1 diabetes. EVs are a composition of biologically active components such as nucleic acids, proteins, metabolites, and lipids that can be transported to particular cells/tissues through the blood system. Through their varied content, EVs can serve as a flexible aid in the diagnosis and management of type 1 diabetes. In this review, we provide an overview of existing knowledge about EVs. We also cover the role of EVs in the pathogenesis, detection, and treatment of type 1 diabetes and the function of EVs in pancreas and islet β-cell transplantation

Extracellular Vesicles in Type 1 Diabetes: A Versatile Tool

Type 1 diabetes is a chronic autoimmune disease affecting nearly 35 million people. This disease develops as T-cells continually attack the β-cells of the islets of Langerhans in the pancreas, which leads to β-cell death, and steadily decreasing secretion of insulin. Lowered levels of insulin minimize the uptake of glucose into cells, thus putting the body in a hyperglycemic state. Despite significant progress in the understanding of the pathophysiology of this disease, there is a need for novel developments in the diagnostics and management of type 1 diabetes. Extracellular vesicles (EVs) are lipid-bound nanoparticles that contain diverse content from their cell of origin and can be used as a biomarker for both the onset of diabetes and transplantation rejection. Furthermore, vesicles can be loaded with therapeutic cargo and delivered in conjunction with a transplant to increase cell survival and long-term outcomes. Crucially, several studies have linked EVs and their cargos to the progression of type 1 diabetes. As a result, gaining a better understanding of EVs would help researchers better comprehend the utility of EVs in regulating and understanding type 1 diabetes. EVs are a composition of biologically active components such as nucleic acids, proteins, metabolites, and lipids that can be transported to particular cells/tissues through the blood system. Through their varied content, EVs can serve as a flexible aid in the diagnosis and management of type 1 diabetes. In this review, we provide an overview of existing knowledge about EVs. We also cover the role of EVs in the pathogenesis, detection, and treatment of type 1 diabetes and the function of EVs in pancreas and islet β-cell transplantation

Mesenchymal Stem Cell-Derived Exosomes: Applications in Regenerative Medicine

Exosomes are a type of extracellular vesicles, produced within multivesicular bodies, that are then released into the extracellular space through a merging of the multivesicular body with the plasma membrane. These vesicles are secreted by almost all cell types to aid in a vast array of cellular functions, including intercellular communication, cell differentiation and proliferation, angiogenesis, stress response, and immune signaling. This ability to contribute to several distinct processes is due to the complexity of exosomes, as they carry a multitude of signaling moieties, including proteins, lipids, cell surface receptors, enzymes, cytokines, transcription factors, and nucleic acids. The favorable biological properties of exosomes including biocompatibility, stability, low toxicity, and proficient exchange of molecular cargos make exosomes prime candidates for tissue engineering and regenerative medicine. Exploring the functions and molecular payloads of exosomes can facilitate tissue regeneration therapies and provide mechanistic insight into paracrine modulation of cellular activities. In this review, we summarize the current knowledge of exosome biogenesis, composition, and isolation methods. We also discuss emerging healing properties of exosomes and exosomal cargos, such as microRNAs, in brain injuries, cardiovascular disease, and COVID-19 amongst others. Overall, this review highlights the burgeoning roles and potential applications of exosomes in regenerative medicine

Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin

Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications

DS_DISC786509 – Supplemental material for Development and Characterization of Quantitative, High-Throughput-Compatible Assays for Proteolytic Degradation of Glucagon

<p>Supplemental material, DS_DISC786509 for Development and Characterization of Quantitative, High-Throughput-Compatible Assays for Proteolytic Degradation of Glucagon by Caitlin N. Suire, Shelley Lane and Malcolm A. Leissring in SLAS Discovery</p

Targeting Insulin-Degrading Enzyme in Insulin Clearance

© 2021 by the authors.Hepatic insulin clearance, a physiological process that in response to nutritional cues clears ~50–80% of circulating insulin, is emerging as an important factor in our understanding of the pathogenesis of type 2 diabetes mellitus (T2DM). Insulin-degrading enzyme (IDE) is a highly conserved Zn2+-metalloprotease that degrades insulin and several other intermediate-size peptides. Both, insulin clearance and IDE activity are reduced in diabetic patients, albeit the cause-effect relationship in humans remains unproven. Because historically IDE has been proposed as the main enzyme involved in insulin degradation, efforts in the development of IDE inhibitors as therapeutics in diabetic patients has attracted attention during the last decades. In this review, we retrace the path from Mirsky’s seminal discovery of IDE to the present, highlighting the pros and cons of the development of IDE inhibitors as a pharmacological approach to treating diabetic patients.This research was funded by the Ministerio de Economía, Industria y Competitividad, grant number SAF2016-77871-C2-2-R to G.P.; Ministerio de Ciencia e Innovación PID2019-110496RB-C22 to G.P.; European Foundation for the Study of Diabetes (European Diabetes Research Programme on New Targets for Type 2 Diabetes supported by MSD-2017) to G.P.; European Foundation for the Study of Diabetes (EFSD) -Novo Nordisk to B.M.; and the US National Institutes of Health (GM115617) to M.A.L. The project leading to these results has received funding from “La Caixa” Foundation, under agreement LCF/PR/PR18/51130007 to G.P. C.M.G.-C. was supported by a fellowship from the Junta de Castilla y León and the European Social Fund (ORDER EDU/574/2018). B.M. was supported by a “Rising Star” fellowship (EFSD-Novo Nordisk)

Hydroxypyridinethione Inhibitors of Human Insulin‐Degrading Enzyme

Insulin-degrading enzyme (IDE) is a human mononuclear Zn2+ -dependent metalloenzyme that is widely regarded as the primary peptidase responsible for insulin degradation. Despite its name, IDE is also critically involved in the hydrolysis of several other disparate peptide hormones, including glucagon, amylin, and the amyloid β-protein. As such, the study of IDE inhibition is highly relevant to deciphering the role of IDE in conditions such as type-2 diabetes mellitus and Alzheimer disease. There have been few reported IDE inhibitors, and of these, inhibitors that directly target the active-site Zn2+ ion have yet to be fully explored. In an effort to discover new, zinc-targeting inhibitors of IDE, a library of ∼350 metal-binding pharmacophores was screened against IDE, resulting in the identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an effective Zn2+ -binding scaffold. Screening a focused library of HOPTO compounds identified 3-sulfonamide derivatives of 1,2-HOPTO as inhibitors of IDE (Ki values of ∼50 μM). Further structure-activity relationship studies yielded several thiophene-sulfonamide HOPTO derivatives with good, broad-spectrum activity against IDE that have the potential to be useful pharmacological tools for future studies of IDE

Peptidic inhibitors of insulin-degrading enzyme with potential for dermatological applications discovered via phage display.

Insulin-degrading enzyme (IDE) is an atypical zinc-metalloendopeptidase that hydrolyzes insulin and other intermediate-sized peptide hormones, many of which are implicated in skin health and wound healing. Pharmacological inhibitors of IDE administered internally have been shown to slow the breakdown of insulin and thereby potentiate insulin action. Given the importance of insulin and other IDE substrates for a variety of dermatological processes, pharmacological inhibitors of IDE suitable for topical applications would be expected to hold significant therapeutic and cosmetic potential. Existing IDE inhibitors, however, are prohibitively expensive, difficult to synthesize and of undetermined toxicity. Here we used phage display to discover novel peptidic inhibitors of IDE, which were subsequently characterized in vitro and in cell culture assays. Among several peptide sequences tested, a cyclic dodecapeptide dubbed P12-3A was found to potently inhibit the degradation of insulin (Ki = 2.5 ± 0.31 μM) and other substrates by IDE, while also being resistant to degradation, stable in biological milieu, and highly selective for IDE. In cell culture, P12-3A was shown to potentiate several insulin-induced processes, including the transcription, translation and secretion of alpha-1 type I collagen in primary murine skin fibroblasts, and the migration of keratinocytes in a scratch wound migration assay. By virtue of its potency, stability, specificity for IDE, low cost of synthesis, and demonstrated ability to potentiate insulin-induced processes involved in wound healing and skin health, P12-3A holds significant therapeutic and cosmetic potential for topical applications

Mice lacking the transcriptional regulator Bhlhe40 have enhanced neuronal excitability and impaired synaptic plasticity in the hippocampus

<div><p>Bhlhe40 is a transcription factor that is highly expressed in the hippocampus; however, its role in neuronal function is not well understood. Here, we used <i>Bhlhe40</i> null mice on a congenic C57Bl6/J background (<i>Bhlhe40</i> KO) to investigate the impact of Bhlhe40 on neuronal excitability and synaptic plasticity in the hippocampus. <i>Bhlhe40</i> KO CA1 neurons had increased miniature excitatory post-synaptic current amplitude and decreased inhibitory post-synaptic current amplitude, indicating CA1 neuronal hyperexcitability. Increased CA1 neuronal excitability was not associated with increased seizure severity as <i>Bhlhe40</i> KO relative to +/+ (WT) control mice injected with the convulsant kainic acid. However, significant reductions in long term potentiation and long term depression at CA1 synapses were observed in <i>Bhlhe40</i> KO mice, indicating impaired hippocampal synaptic plasticity. Behavioral testing for spatial learning and memory on the Morris Water Maze (MWM) revealed that while <i>Bhlhe40</i> KO mice performed similarly to WT controls initially, when the hidden platform was moved to the opposite quadrant <i>Bhlhe40</i> KO mice showed impairments in relearning, consistent with decreased hippocampal synaptic plasticity. To investigate possible mechanisms for increased neuronal excitability and decreased synaptic plasticity, a whole genome mRNA expression profile of <i>Bhlhe40</i> KO hippocampus was performed followed by a chromatin immunoprecipitation sequencing (ChIP-Seq) screen of the validated candidate genes for Bhlhe40 protein-DNA interactions consistent with transcriptional regulation. Of the validated genes identified from mRNA expression analysis, insulin degrading enzyme (<i>Ide</i>) had the most significantly altered expression in hippocampus and was significantly downregulated on the RNA and protein levels; although Bhlhe40 did not occupy the <i>Ide</i> gene by ChIP-Seq. Together, these findings support a role for Bhlhe40 in regulating neuronal excitability and synaptic plasticity in the hippocampus and that indirect regulation of <i>Ide</i> transcription may be involved in these phenotypes.</p></div