Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

Structure-Guided Design of N-Methylpropargylamino-Quinazoline Derivatives as Multipotent Agents for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.

Validation and Update of the Lémann Index to Measure Cumulative Structural Bowel Damage in Crohn's Disease.

BACKGROUND & AIMS: The Lémann Index is a tool measuring cumulative structural bowel damage in Crohn's disease (CD). We reported on its validation and updating. METHODS: This was an international, multicenter, prospective, cross-sectional observational study. At each center, 10 inclusions, stratified by CD duration and location, were planned. For each patient, the digestive tract was divided into 4 organs, upper tract, small bowel, colon/rectum, anus, and subsequently into segments, explored systematically by magnetic resonance imaging and by endoscopies in relation to disease location. For each segment, investigators retrieved information on previous surgical procedures, identified predefined strictures and penetrating lesions of maximal severity (grades 1-3) at each organ investigational method (gastroenterologist and radiologist for magnetic resonance imaging), provided segmental damage evaluation ranging from 0.0 to 10.0 (complete resection). Organ resection-free cumulative damage evaluation was then calculated from the sum of segmental damages. Then investigators provided a 0-10 global damage evaluation from the 4-organ standardized cumulative damage evaluations. Simple linear regressions of investigator damage evaluations on their corresponding Lémann Index were studied, as well as calibration plots. Finally, updated Lémann Index was derived through multiple linear mixed models applied to combined development and validation samples. RESULTS: In 15 centers, 134 patients were included. Correlation coefficients between investigator damage evaluations and Lémann Indexes were >0.80. When analyzing data in 272 patients from both samples and 27 centers, the unbiased correlation estimates were 0.89, 0,97, 0,94, 0.81, and 0.91 for the 4 organs and globally, and stable when applied to one sample or the other. CONCLUSIONS: The updated Lémann Index is a well-established index to assess cumulative bowel damage in CD that can be used in epidemiological studies and disease modification trials.

Follistatin-like 1 and its paralogs in heart development and cardiovascular disease.

Cardiovascular diseases (CVDs) are a group of disorders affecting the heart and blood vessels and a leading cause of death worldwide. Thus, there is a need to identify new cardiokines that may protect the heart from damage as reported in GBD 2017 Causes of Death Collaborators (2018) (The Lancet 392:1736-1788). Follistatin-like 1 (FSTL1) is a cardiokine that is highly expressed in the heart and released to the serum after cardiac injury where it is associated with CVD and predicts poor outcome. The action of FSTL1 likely depends not only on the tissue source but also post-translation modifications that are target tissue- and cell-specific. Animal studies examining the effect of FSTL1 in various models of heart disease have exploded over the past 15 years and primarily report a protective effect spanning from inhibiting inflammation via transforming growth factor, preventing remodeling and fibrosis to promoting angiogenesis and hypertrophy. A better understanding of FSTL1 and its homologs is needed to determine whether this protein could be a useful novel biomarker to predict poor outcome and death and whether it has therapeutic potential. The aim of this review is to provide a comprehensive description of the literature for this family of proteins in order to better understand their role in normal physiology and CVD.

Variants in the MIPEP gene presenting with complex neurological phenotype without cardiomyopathy, impair OXPHOS protein maturation and lead to a reduced OXPHOS abundance in patient cells.

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants-one was not previously linked to mitochondrial disease-who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells.

Familial occurrence of skeletal developmental anomalies as a reflection of biological relationships in a genealogically documented Central European sample (19th to 20th centuries).

Skeletal developmental anomalies (SDA) are a subject of constant interest across scientific disciplines, but still mostly as isolates and curiosities. The aim of this study was to find out to what extent the occurrence of SDA reflects documented biological relationships. The skeletal remains of 34 individuals with known genealogical data were available, members of one family over four generations (19th to 20th centuries, Bohemia, Czech Republic), including some inbred individuals. The occurrence of 89 SDA was assessed on the basis of scopic morphological evaluation and X-ray and CT examinations. The degree of similarity between individuals was calculated using a "similarity coefficient" (SC). A linear model was used to test the relationship between positive values of the SC and the relatedness of biologically related individuals. Simultaneously, based on population frequencies of the evaluated anomalies, those that could be considered familial were recorded. A statistically significant relationship between morphological similarity and the biological distance between individuals was found. The greatest similarity was found among close relatives such as parents and children, siblings, or grandparents and grandchildren. The effect of increased consanguinity on the occurrence of anomalies was not confirmed, however. Seventeen SDA shared by closely related individuals were found in the sample, supporting the documented family relationships among them. Eleven of these were selected as possibly familial, but only five were statistically significant: an elongated styloid process, a cervical block vertebrae (arch, facet joints), hamate hamulus aplasia, anteater nose sign, and incomplete fusion of the S1 spinous process. There were also 28 cases of individual occurrences of 17 different SDA, without connection to the documented relationships between individuals.

N-linked glycosylation of the mGlu7 receptor regulates the forward trafficking and transsynaptic interaction with Elfn1.

Metabotropic glutamate receptor 7 (mGlu7) regulates neurotransmitter release at the presynaptic active zone in the mammalian brain. The regulation of mGlu7 trafficking into and out of the plasma membrane by binding proteins within the C-terminal region of mGlu7 governs the bidirectional synaptic plasticity. However, the functional importance of the extracellular domain of mGlu7 has not yet been characterized. N-glycosylation is an abundant posttranslational modification that plays crucial roles in protein folding and forward trafficking, but the role of N-glycosylation in mGlu7 function remains unknown. In this study, we find that mGlu7 is N-glycosylated at four asparagine residues in heterologous cells and rat cultured neurons. We demonstrate that N-glycosylation is essential for forward transport and surface expression of mGlu7. Deglycosylated mGlu7 is retained in the ER, obstructing expression on the cell surface, and is degraded through the autophagolysosomal degradation pathway. In addition, we identify the binding domain of mGlu7 to Elfn1, a transsynaptic adhesion protein. We find that N-glycosylation of mGlu7 promotes its interaction with Elfn1, thereby enabling proper localization and stable surface expression of mGlu7 at the presynaptic active zone. These findings provide evidence that N-glycans act to modulate the surface expression, stability, and function of mGlu7.

Sex estimation using external morphology of the frontal bone and frontal sinuses in a contemporary Czech population.

Sex estimation is a task of utmost importance in forensic anthropology and bioarcheology. Along with the pelvic bone, the skull is the most important source of sexual dimorphism. On the human skull, the upper third of the face (i.e., the frontal bone) is one of the most significant sexually dimorphic structures useful in anthropological research, especially when studied by methods of virtual anthropology. This study was focused on sex estimation using the form and shape of the external surface of the frontal bone with or without the inclusion of its sinuses. The study sample consisted of 103 cranial CT images from a contemporary Czech population. Three-dimensional virtual models of the frontal bones and sinuses were analyzed using geometric morphometrics and multidimensional statistics: coherent point drift-dense correspondence analysis (CPD-DCA), principal component analysis (PCA), and support vector machine (SVM). The whole external frontal surface was significantly different between males and females both in form and shape. The greatest total success rate of sex estimation based on form was 93.2%, which decreased to 86.41% after crossvalidation, and this model identified females and males with the same accuracy. The best estimation based on shape reached a success rate of 91.26%, with slightly greater accuracy for females. After crossvalidation, however, the success rate decreased to 83.49%. The differences between sexes were significant also in the volume and surface of the frontal sinuses, but the sex estimation had only 64.07% accuracy after crossvalidation. Simultaneous use of the shape of the frontal surface and the frontal sinuses improved the total success rate to 98.05%, which decreased to 84.46% after crossvalidation.

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives.

Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action. SIGNIFICANCE STATEMENT: Synaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

Muscle-specific microRNAs in skeletal muscle development.

Proper muscle function constitutes a precondition for good heath and an active lifestyle during an individual's lifespan and any deviations from normal skeletal muscle development and its functions may lead to numerous health conditions including e.g. myopathies and increased mortality. It is thus not surprising that there is an increasing need for understanding skeletal muscle developmental processes and the associated molecular pathways, especially as such information could find further uses in therapy. The understanding of complex skeletal muscle developmental networks was broadened with the discovery of microRNA (miRNA) molecules. MicroRNAs are evolutionary conserved small non-coding RNAs capable of negatively regulating gene expression on a post-transcriptional level by means of miRNA-mRNA interaction. Several miRNAs expressed exclusively in muscle have been labeled myomiRs. MyomiRs represent an integral part of skeletal muscle development, i.e. playing a significant role during skeletal muscle proliferation, differentiation and regeneration. The purpose of this review is to provide a summary of current knowledge regarding the involvement of myomiRs in the individual phases of myogenesis and other aspects of skeletal muscle biology, along with an up-to-date list of myomiR target genes and their functions in skeletal muscle and miRNA-related therapeutic approaches and future prospects.

Multi-target-directed therapeutic potential of 7-methoxytacrine-adamantylamine heterodimers in the Alzheimer's disease treatment.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and currently there is no efficient treatment. The classic drug-design strategy based on the "one-molecule-one-target" paradigm was found to be ineffective in the case of multifactorial diseases like AD. A novel multi-target-directed ligand strategy based on the assumption that a single compound consisting of two or more distinct pharmacophores is able to hit multiple targets has been proposed as promising. Herein, we investigated 7-methoxytacrine - memantine heterodimers developed with respect to the multi-target-directed ligand theory. The spectroscopic, microscopic and cell culture methods were used for systematic investigation of the interference of the heterodimers with ß-secretase (BACE1) activity, Aß peptide amyloid fibrillization (amyloid theory) and interaction with M1 subtype of muscarinic (mAChRs), nicotinic (nAChRs) acetylcholine receptors (cholinergic theory) and N-methyl-d-aspartate receptors (NMDA) (glutamatergic theory). The drug-like properties of selected compounds have been evaluated from the point of view of blood-brain barrier penetration and cell proliferation. We have confirmed the multipotent effect of novel series of compounds. They inhibited effectively Aß peptide amyloid fibrillization and affected the BACE1 activity. Moreover, they have AChE inhibitory potency but they could not potentiate cholinergic transmission via direct interaction with cholinergic receptors. All compounds were reported to act as an antagonist of both M1 muscarinic and muscle-type nicotinic receptors. We have found that 7-methoxytacrine - memantine heterodimers are able to hit multiple targets associated with Alzheimer's disease and thus, have a potential clinical impact for slowing or blocking the neurodegenerative process related to this disease.

Calculation of nonlinearly deformed membrane shape of liquid lens caused by uniform pressure.

The paper discusses a numerical calculation of deformation of a circular axisymmetric membrane of a liquid lens caused by the pressure of an optical liquid. Since such deflections of the membrane are many times larger than the membrane thickness, a nonlinear model is applied and generalized relationships are derived that characterize the resulting shape with a high precision and permit an accurate analysis of imaging properties of the lens and of optical aberrations. By comparison with experimental data, it is shown that the presented model is suitable to describe the deformation of the membrane of the lens.

Deformation of a prestressed liquid lens membrane.

This paper presents a complete model for analysis of the deformed shape of a prestressed circular axisymmetric membrane of a liquid lens. The governing equations are derived using the exact relation between displacements and the Green-Lagrange strains combined with the Saint Venant-Kirchhoff material law, which postulates a linear relation between the Green-Lagrange strains and the second Piola-Kirchoff stresses. A numerical solution based on minimization of potential energy is illustrated by an example, and the dependence of the maximum membrane deflection on material properties and initial prestress is analyzed. The theoretical model is then experimentally validated. It is shown that the model is suitable for large-strain analysis of liquid lens membranes and provides sufficiently accurate results that can be used in further analyses and simulations of imaging properties of active optical elements based on liquid lenses.

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum.

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.

Biochemical and electrophysiological characterization of N-glycans on NMDA receptor subunits.

In mammals, excitatory synapses contain two major types of ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors (NMDARs). Both receptor types are comprised of several subunits that are post-translationally modified by N-glycosylation. However, the precise N-glycans that are attached to these receptor types are largely unknown. Here, we used biochemistry to confirm that native NMDARs are extensively N-glycosylated; moreover, we found that the NMDAR GluN2B subunit differs from GluN1 subunits with respect to endoglycosidase H sensitivity. Next, we used a complete panel of lectins to determine the glycan composition of NMDARs in both cerebellar tissue and cultured cerebellar granule cells. Our experiments identified 23 lectins that pulled down both the GluN1 and GluN2B NMDAR subunits. We then performed an electrophysiological analysis using representative lectins and found that pre-incubating cerebellar granule cells with the AAL, WGA, or ConA alters the receptor's biophysical properties; this lectin-mediated effect was eliminated when the cells were deglycosylated with peptide-N-glycosidase F. Similar lectin-mediated effects were observed using HEK293 cells that express recombinant GluN1/GluN2B receptors. Finally, using mutant recombinant GluN subunits expressed in HEK293 cells, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data provide new insight into the role that N-glycosylation plays in regulating the function of NMDA receptors in the central nervous system. All animal experiments were performed in accordance with relevant institutional ethics guidelines and regulations with respect to protecting animal welfare. We examined the N-glycan composition of NMDA receptors (NMDARs) using deglycosylating enzymes, lectin-based biochemistry, and electrophysiology. Our results revealed that cerebellar NMDARs associate with 23 different lectins that have unique specificities for glycan structures. Furthermore, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data shed light on the glycan composition of NMDARs, revealing potential targets for the development of novel therapeutic approaches.

Cholesterol modulates open probability and desensitization of NMDA receptors.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-ß-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-ß-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

mTOR pathway inhibition alters proliferation as well as differentiation of neural stem cells.

Introduction: Neural stem cells (NSCs) are essential for both embryonic development and adult neurogenesis, and their dysregulation causes a number of neurodevelopmental disorders, such as epilepsy and autism spectrum disorders. NSC proliferation and differentiation in the developing brain is a complex process controlled by various intrinsic and extrinsic stimuli. The mammalian target of rapamycin (mTOR) regulates proliferation and differentiation, among other cellular functions, and disruption in the mTOR pathway can lead to severe nervous system development deficits. In this study, we investigated the effect of inhibition of the mTOR pathway by rapamycin (Rapa) on NSC proliferation and differentiation. Methods: The NSC cultures were treated with Rapa for 1, 2, 6, 24, and 48 h. The effect on cellular functions was assessed by immunofluorescence staining, western blotting, and proliferation/metabolic assays. Results: mTOR inhibition suppressed NSC proliferation/metabolic activity as well as S-Phase entry by as early as 1 h of Rapa treatment and this effect persisted up to 48 h of Rapa treatment. In a separate experiment, NSCs were differentiated for 2 weeks after treatment with Rapa for 24 or 48 h. Regarding the effect on neuronal and glial differentiation (2 weeks post-treatment), this was suppressed in NSCs deficient in mTOR signaling, as evidenced by downregulated expression of NeuN, MAP2, and GFAP. We assume that the prolonged effect of mTOR inhibition is realized due to the effect on cytoskeletal proteins. Discussion: Here, we demonstrate for the first time that the mTOR pathway not only regulates NSC proliferation but also plays an important role in NSC differentiation into both neuronal and glial lineages.

Pro-cognitive effects of dual tacrine derivatives acting as cholinesterase inhibitors and NMDA receptor antagonists.

Therapeutic options for Alzheimer's disease are limited. Dual compounds targeting two pathways concurrently may enable enhanced effect. The study focuses on tacrine derivatives inhibiting acetylcholinesterase (AChE) and simultaneously N-methyl-D-aspartate (NMDA) receptors. Compounds with balanced inhibitory potencies for the target proteins (K1578 and K1599) or increased potency for AChE (K1592 and K1594) were studied to identify the most promising pro-cognitive compound. Their effects were studied in cholinergic (scopolamine-induced) and glutamatergic (MK-801-induced) rat models of cognitive deficits in the Morris water maze. Moreover, the impacts on locomotion in the open field and AChE activity in relevant brain structures were investigated. The effect of the most promising compound on NMDA receptors was explored by in vitro electrophysiology. The cholinergic antagonist scopolamine induced a deficit in memory acquisition, however, it was unaffected by the compounds, and a deficit in reversal learning that was alleviated by K1578 and K1599. K1578 and K1599 significantly inhibited AChE in the striatum, potentially explaining the behavioral observations. The glutamatergic antagonist dizocilpine (MK-801) induced a deficit in memory acquisition, which was alleviated by K1599. K1599 also mitigated the MK-801-induced hyperlocomotion in the open field. In vitro patch-clamp corroborated the K1599-associated NMDA receptor inhibitory effect. K1599 emerged as the most promising compound, demonstrating pro-cognitive efficacy in both models, consistent with intended dual effect. We conclude that tacrine has the potential for development of derivatives with dual in vivo effects. Our findings contributed to the elucidation of the structural and functional properties of tacrine derivatives associated with optimal in vivo pro-cognitive efficacy.

Morphing cholinesterase inhibitor amiridine into multipotent drugs for the treatment of Alzheimer's disease.

The search for novel drugs to address the medical needs of Alzheimer's disease (AD) is an ongoing process relying on the discovery of disease-modifying agents. Given the complexity of the disease, such an aim can be pursued by developing so-called multi-target directed ligands (MTDLs) that will impact the disease pathophysiology more comprehensively. Herewith, we contemplated the therapeutic efficacy of an amiridine drug acting as a cholinesterase inhibitor by converting it into a novel class of novel MTDLs. Applying the linking approach, we have paired amiridine as a core building block with memantine/adamantylamine, trolox, and substituted benzothiazole moieties to generate novel MTDLs endowed with additional properties like N-methyl-d-aspartate (NMDA) receptor affinity, antioxidant capacity, and anti-amyloid properties, respectively. The top-ranked amiridine-based compound 5d was also inspected by in silico to reveal the butyrylcholinesterase binding differences with its close structural analogue 5b. Our study provides insight into the discovery of novel amiridine-based drugs by broadening their target-engaged profile from cholinesterase inhibitors towards MTDLs with potential implications in AD therapy.