Calcineurin controls expression of EAAT1/GLAST in mouse and human cultured astrocytes through dynamic regulation of protein synthesis and degradation

Alterations in the expression of glutamate/aspartate transporter (GLAST) have been associated with several neuropathological conditions including Alzheimer\u2019s disease and epilepsy. However, the mechanisms by which GLAST expression is altered are poorly understood. Here we used a combination of pharmacological and genetic approaches coupled with quantitative PCR and Western blot to investigate the mechanism of the regulation of GLAST expression by a Ca2+ /calmodulin-activated phosphatase calcineurin (CaN). We show that treatment of cultured hippocampal mouse and fetal human astrocytes with a CaN inhibitor FK506 resulted in a dynamic modulation of GLAST protein expression, being downregulated after 24\u201348 h, but upregulated after 7 days of continuous FK506 (200 nM) treatment. Protein synthesis, as assessed by puromycin incorporation in neo-synthesized polypeptides, was inhibited already after 1 h of FK506 treatment, while the use of a proteasome inhibitor MG132 (1 \ub5M) shows that GLAST protein degradation was only suppressed after 7 days of FK506 treatment. In astrocytes with constitutive genetic ablation of CaN both protein synthesis and degradation were significantly inhibited. Taken together, our data suggest that, in cultured astrocytes, CaN controls GLAST expression at a posttranscriptional level through regulation of GLAST protein synthesis and degradation

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco’s Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38–24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer’s disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines. © 2023 by the authors

A tetracationic porphyrin with dual anti-prion activity

Prions are deadly infectious agents made of PrPSc, a misfolded variant of the cellular prion protein (PrPC) which self-propagates by inducing misfolding of native PrPC. PrPSc can adopt different pathogenic conformations (prion strains), which can be resistant to potential drugs, or acquire drug resistance, hampering the development of effective therapies. We identified Zn(II)-BnPyP, a tetracationic porphyrin that binds to distinct domains of native PrPC, eliciting a dual anti-prion effect. Zn(II)-BnPyP binding to a C-terminal pocket destabilizes the native PrPC fold, hindering conversion to PrPSc; Zn(II)-BnPyP binding to the flexible N-terminal tail disrupts N- to C-terminal interactions, triggering PrPC endocytosis and lysosomal degradation, thus reducing the substrate for PrPSc generation. Zn(II)-BnPyP inhibits propagation of different prion strains in vitro, in neuronal cells and organotypic brain cultures. These results identify a PrPC-targeting compound with an unprecedented dual mechanism of action which might be exploited to achieve anti-prion effects without engendering drug resistance

Immunopurification of Pathological Prion Protein Aggregates

Background: Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP molecules. A great deal of effort has been devoted to developing protocols for purifying PrP for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases. Principal Findings: We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP and mutant PrP aggregates at electrophoretic homogeneity. PrP purified from prion-infected mice was able to seed misfolding of PrP in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons. Significance: The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates

Dual effects of 9-cis retinoic acid on ACTH-dependent hyperplastic adrenal tissues

Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing\u2019s syndrome. Adrenal specimens from six patients with Cushing\u2019s disease were incubated with 10\ua0nM\u20131\ua0\ub5M 9-cis retinoic acid with and without 10\ua0nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10\u2013100\ua0nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing\u2019s disease

The hydrophobic core region governs mutant prion protein aggregation and intracellular retention

Approx. 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to mutations in the gene encoding PrP (prion protein), a GPI (glycosylphosphatidylinositol)-anchored protein whose function is not clear. The cellular mechanisms by which PrP mutations cause disease are also not known. Soon after synthesis in the ER (endoplasmic reticulum), several mutant PrPs misfold and become resistant to phospholipase cleavage of their GPI anchor. The biosynthetic maturation of the misfoldedmolecules in the ER is delayed and, during transit in the secretory pathway, they form detergent-insoluble and protease-resistant aggregates, suggesting that intracellular PrP aggregation may play a pathogenic role. We have investigated the consequence of deleting residues 114-121 within the hydrophobic core of PrP on the aggregation and cellular localization of two pathogenic mutants that accumulate in the ER and Golgi apparatus. Compared with their full-length counterparts, the deleted molecules formed smaller protease-sensitive aggregates and were more efficiently transported to the cell surface and released by phospholipase cleavage. These results indicate that mutant PrP aggregation and intracellular retention are closely related and depend critically on the integrity of the hydrophobic core. The discovery that \u394114-121 counteracts misfolding and improves the cellular trafficking of mutant PrP provides an unprecedented model for assessing the role of intracellular aggregation in the pathogenesis of prion diseases. \ua9 The Authors

Effect of retinoic acid on human adrenal corticosteroid synthesis

Aims Retinoic acid has recently yielded promising results in the treatment of Cushing's disease, i.e., excess cortisol secretion due to a pituitary corticotropin (ACTH)-secreting adenoma. In addition to its effect on the tumoral corticotrope cell, clinical results suggest an additional adrenal site of action. Aim of this study was to evaluate whether retinoic acid modulates cortisol synthesis and secretion by human adrenals in vitro. Main methods Primary cultures from 10 human adrenals specimens were incubated with 10 nM, 100 nM and 1 \u3bcM retinoic acid with and without 10 nM ACTH for 24 h. Cortisol levels were measured by radioimmunoassay and CYP11A1, STAR and MC2R gene expression analyzed by real-time PCR. Key findings Retinoic acid increased cortisol secretion (149.5 \ub1 33.01%, 151.3 \ub1 49.45% and 129.3 \ub1 8.32% control secretion for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05) and potentiated STAR expression (1.51 \ub1 0.22, 1.56 \ub1 0.15 and 1.59 \ub1 0.14 fold change over baseline, for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05). Concurrently, retinoic acid markedly blunted constitutional and ACTH-induced MC2R expression (0.66 \ub1 0.11, 0.62 \ub1 0.08 and 0.53 \ub1 0.07 fold change over baseline, for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05; 0.71 \ub1 0.10, 0.51 \ub1 0.07 and 0.51 \ub1 0.08 fold change over ACTH alone, for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05). No effect on CYP11A1 was observed. Significance Retinoic acid stimulates cortisol synthesis and secretion in human adrenals and at the same time markedly blunts ACTH receptor transcription. These results reveal a novel, adrenal effect of retinoic acid which may contribute to its efficacy in patients with Cushing's disease

Epitope scanning indicates structural differences in brain-derived monomeric and aggregated mutant prion proteins related to genetic prion diseases

Genetic Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia and prion protein cerebral amyloid angiopathy are clinically and neuropathologically distinct neurodegenerative diseases linked to mutations in the PRNP gene encoding the cellular prion protein (PrPC). How sequence variants of PRNP encode the information to specify these disease phenotypes is not known. It is suggested that each mutation produces a misfolded variant of PrPC with specific neurotoxic properties. However, structural studies of recombinant PrP did not detect major differences between wild-type andmutant molecules, pointing to the importance of investigatingmutant PrPs from mammalian brains. We used surface plasmon resonance and a slot-blot immunoassay to analyse the antibody-binding profiles of soluble and insoluble PrP molecules extracted from the brains of transgenic mice modelling different prion diseases. By measuring the reactivity of monoclonal antibodies against different PrP epitopes, we obtained evidence of conformational differences between wild-type and mutant PrPs, and among different mutants. We detected structural heterogeneity in both monomeric and aggregated PrP, supporting the hypothesis that the phenotype of genetic prion diseases is encoded by mutant PrP conformation and assembly state. © 2013 Biochemical Society

Effect of retinoic acid on human adrenal corticosteroid synthesis

Aims Retinoic acid has recently yielded promising results in the treatment of Cushing's disease, i.e., excess cortisol secretion due to a pituitary corticotropin (ACTH)-secreting adenoma. In addition to its effect on the tumoral corticotrope cell, clinical results suggest an additional adrenal site of action. Aim of this study was to evaluate whether retinoic acid modulates cortisol synthesis and secretion by human adrenals in vitro. Main methods Primary cultures from 10 human adrenals specimens were incubated with 10 nM, 100 nM and 1 μM retinoic acid with and without 10 nM ACTH for 24 h. Cortisol levels were measured by radioimmunoassay and CYP11A1, STAR and MC2R gene expression analyzed by real-time PCR. Key findings Retinoic acid increased cortisol secretion (149.5 ± 33.01%, 151.3 ± 49.45% and 129.3 ± 8.32% control secretion for 10 nM, 100 nM and 1 μM respectively, p < 0.05) and potentiated STAR expression (1.51 ± 0.22, 1.56 ± 0.15 and 1.59 ± 0.14 fold change over baseline, for 10 nM, 100 nM and 1 μM respectively, p < 0.05). Concurrently, retinoic acid markedly blunted constitutional and ACTH-induced MC2R expression (0.66 ± 0.11, 0.62 ± 0.08 and 0.53 ± 0.07 fold change over baseline, for 10 nM, 100 nM and 1 μM respectively, p < 0.05; 0.71 ± 0.10, 0.51 ± 0.07 and 0.51 ± 0.08 fold change over ACTH alone, for 10 nM, 100 nM and 1 μM respectively, p < 0.05). No effect on CYP11A1 was observed. Significance Retinoic acid stimulates cortisol synthesis and secretion in human adrenals and at the same time markedly blunts ACTH receptor transcription. These results reveal a novel, adrenal effect of retinoic acid which may contribute to its efficacy in patients with Cushing's disease