Multitarget drugs as potential therapeutic agents for alzheimer's disease. A new family of 5-substituted indazole derivatives as cholinergic and BACE1 inhibitors.

Multitarget drugs are a promising therapeutic approach against Alzheimer's disease. In this work, a new family of 5-substituted indazole derivatives with a multitarget profile including cholinesterase and BACE1 inhibition is described. Thus, the synthesis and evaluation of a new class of 5-substituted indazoles has been performed. Pharmacological evaluation includes in vitro inhibitory assays on AChE/BuChE and BACE1 enzymes. Also, the corresponding competition studies on BuChE were carried out. Additionally, antioxidant properties have been calculated from ORAC assays. Furthermore, studies of anti-inflammatory properties on Raw 264.7 cells and neuroprotective effects in human neuroblastoma SH-SY5Y cells have been performed. The results of pharmacological tests have shown that some of these 5-substituted indazole derivatives 1-4 and 6 behave as AChE/BuChE and BACE1 inhibitors, simultaneously. In addition, some indazole derivatives showed anti-inflammatory (3, 6) and neuroprotective (1-4 and 6) effects against Aß-induced cell death in human neuroblastoma SH-SY5Y cells with antioxidant properties.

Increased YKL-40 but Not C-Reactive Protein Levels in Patients with Alzheimer's Disease.

Neuroinflammation is a common feature in Alzheimer's (AD) and Parkinson's (PD) disease. In the last few decades, a testable hypothesis was proposed that protein-unfolding events might occur due to neuroinflammatory cascades involving alterations in the crosstalk between glial cells and neurons. Here, we tried to clarify the pattern of two of the most promising biomarkers of neuroinflammation in cerebrospinal fluid (CSF) in AD and PD. This study included cognitively unimpaired elderly patients, patients with mild cognitive impairment, patients with AD dementia, and patients with PD. CSF samples were analyzed for YKL-40 and C-reactive protein (CRP). We found that CSF YKL-40 levels were significantly increased only in dementia stages of AD. Additionally, increased YKL-40 levels were found in the cerebral orbitofrontal cortex from AD patients in agreement with augmented astrogliosis. Our study confirms that these biomarkers of neuroinflammation are differently detected in CSF from AD and PD patients.

Retinal Proteomics of a Mouse Model of Dystroglycanopathies Reveals Molecular Alterations in Photoreceptors.

Mutations in the POMT1 gene, encoding a protein O-mannosyltransferase essential for α-dystroglycan (α-DG) glycosylation, are frequently observed in a group of rare congenital muscular dystrophies, collectively known as dystroglycanopathies. However, it is hitherto unclear whether the effects seen in affected patients can be fully ascribed to α-DG hypoglycosylation. To study this, here we used comparative mass spectrometry-based proteomics and immunofluorescence microscopy and investigated the changes in the retina of mice in which Pomt1 is specifically knocked out in photoreceptor cells. Our results demonstrate significant proteomic changes and associated structural alteration in photoreceptor cells of Pomt1 cKO mice. In addition to the effects related to impaired α-DG O-mannosylation, we observed morphological alterations in the outer segment that are associated with dysregulation of a relatively understudied POMT1 substrate (KIAA1549), BBSome proteins, and retinal stress markers. In conclusion, our study provides new hypotheses to explain the phenotypic changes that are observed in the retina of patients with dystroglycanopathies.

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents.

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.

Endothelial-specific deficiency of megalin in the brain protects mice against high-fat diet challenge.

BACKGROUND: The increasing risk of obesity and diabetes among other metabolic disorders are the consequence of shifts in dietary patterns with high caloric-content food intake. We previously reported that megalin regulates energy homeostasis using blood-brain barrier (BBB) endothelial megalin-deficient (EMD) mice, since these animals developed obesity and metabolic syndrome upon normal chow diet administration. Obesity in mid-life appears to be related to greater dementia risk and represents an increasing global health issue. We demonstrated that EMD phenotype induced impaired learning ability and recognition memory, neurodegeneration, neuroinflammation, reduced neurogenesis, and mitochondrial deregulation associated with higher mitochondrial mass in cortical tissues. METHODS: EMD mice were subjected to normal chow and high-fat diet (HFD) for 14 weeks and metabolic changes were evaluated. RESULTS: Surprisingly, BBB megalin deficiency protected against HFD-induced obesity improving glucose tolerance and preventing hepatic steatosis. Compared to wild type (wt), the brain cortex in EMD mice showed increased levels of the mitochondrial biogenesis regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and uncoupling protein 2 (UCP2), a thermogenic protein involved in the regulation of energy metabolism. This agreed with the previously found increased mitochondrial mass in the transgenic mice. Upon HFD challenge, we demonstrated these two proteins were found elevated in wt mice but reported no changes over the already increased levels in EMD animals. CONCLUSION: We propose a protective role for megalin on diet-induce obesity, suggesting this could be related to metabolic disturbances found in dementia through brain endocrine system communications.

Impairment of photoreceptor ribbon synapses in a novel Pomt1 conditional knockout mouse model of dystroglycanopathy.

Hypoglycosylation of α-dystroglycan (α-DG) resulting from deficiency of protein O-mannosyltransferase 1 (POMT1) may cause severe neuromuscular dystrophies with brain and eye anomalies, named dystroglycanopathies. The retinal involvement of these disorders motivated us to generate a conditional knockout (cKO) mouse experiencing a Pomt1 intragenic deletion (exons 3-4) during the development of photoreceptors, mediated by the Cre recombinase expressed from the cone-rod homeobox (Crx) gene promoter. In this mouse, retinal α-DG was unglycosylated and incapable of binding laminin. Retinal POMT1 deficiency caused significant impairments in both electroretinographic recordings and optokinetic reflex in Pomt1 cKO mice, and immunohistochemical analyses revealed the absence of ß-DG and of the α-DG-interacting protein, pikachurin, in the outer plexiform layer (OPL). At the ultrastructural level, noticeable alterations were observed in the ribbon synapses established between photoreceptors and bipolar cells. Therefore, O-mannosylation of α-DG in the retina carried out by POMT1 is crucial for the establishment of proper synapses at the OPL and transmission of visual information from cones and rods to their postsynaptic neurons.

Clinical features and molecular characterization of a patient with muscle-eye-brain disease: a novel mutation in the POMGNT1 gene.

Muscle-eye-brain disease is a congenital muscular dystrophy characterized by structural brain and eye defects. Here, we describe a 12-year-old boy with partial agenesis of corpus callosum, ventriculomegaly, flattened brain stem, diffuse pachygyria, blindness, profound cognitive deficiencies, and generalized muscle weakness, yet without a clear dystrophic pattern on muscle biopsy. There was no glycosylation of α-dystroglycan and the genetic screening revealed a novel truncating mutation, c.1545delC (p.Tyr516Thrfs*21), and a previously identified missense mutation, c.1469G>A (p.Cys490Tyr), in the protein O-mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) gene. These findings broaden the clinical spectrum of muscle-eye-brain disease to include pronounced hypotonia with severe brain and eye malformations, yet with mild histopathologic changes in the muscle specimen, despite the absence of glycosylated α-dystroglycan.