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OBJECTIVE: This study introduces a novel desktop micro-CT scanner designed for dynamic perfusion imaging in mice, aimed at enhancing preclinical imaging capabilities with high resolution and low radiation doses. Approach: The micro-CT system features a custom-built rotating table capable of both circular and helical scans, enabled by a small-bore slip ring for continuous rotation. Images were reconstructed with a temporal resolution of 3.125 seconds and an isotropic voxel size of 65 µm, with potential for higher resolution scanning. The system's static performance was validated using standard quality assurance phantoms. Dynamic performance was assessed with a custom 3D-bioprinted tissue-mimetic phantom simulating single-compartment vascular flow. Flow measurements ranged from 1.5 mL/min to 9 mL/min, with perfusion metrics such as time-to-peak (TTP), mean transit time (MTT), and blood flow index (BFI) calculated. In vivo experiments involved mice with different genetic risk factors for Alzheimer's and cardiovascular diseases to showcase the system's capabilities for perfusion imaging. Main Results: The static performance validation confirmed that the system meets standard quality metrics, such as spatial resolution and uniformity. The dynamic evaluation with the 3D-bioprinted phantom demonstrated linearity in hemodynamic flow measurements and effective quantification of perfusion metrics. In vivo experiments highlighted the system's potential to capture detailed perfusion maps of the brain, lungs, and kidneys. The observed differences in perfusion characteristics between genotypic mice illustrated the system's capability to detect physiological variations, though the small sample size precludes definitive conclusions. Significance: The turn-table micro-CT system represents a significant advancement in preclinical imaging, providing high-resolution, low-dose dynamic imaging for a range of biological and medical research applications. Future work will focus on improving temporal resolution, expanding spectral capabilities, and integrating deep learning techniques for enhanced image reconstruction and analysis.
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In this study, we explored the impact of caregiving on quality of life and health perceptions and outlined the profile of grandparent caregivers in Andalusia (Spain) in terms of a range of sociodemographic variables related to the care of their grandchildren. A sample of 171 participants (21.6% men) completed the Health-Related Quality of Life (HRQoL) questionnaire and another ad hoc one providing sociodemographic and caregiving data. We studied the relationships between these variables and HRQoL using ANOVA, chi-square and Multiple Linear Regression. We found a mainly female profile for the care of grandchildren and interesting relationships for the physical and mental components of HRQoL. Some relationships were marked by gender: caregiving for pleasure was more often the motive for men while by imposition was more common among women. We discuss the impact of caregiving on health according to the Self-Determination Theory and suggest practical implications derived from the main findings.
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Amyotrophic lateral sclerosis is a fatal neurodegenerative disease without a cure to reverse its progression. Its main hallmark is the nuclear protein TDP-43, which undergoes different post-translational modifications leading to a loss of function in the nucleus and an increase in toxicity in the cytoplasm. Previous reports have indicated that pathogenic TDP-43 exhibits prion-like propagation in various contexts. With the aim of advancing therapeutics focused on preventing the propagation of TDP-43 pathology, we studied the potential role of pathogenic TDP-43 in lymphoblasts from sporadic ALS patients. We used lymphoblastoid cell lines from sporadic ALS patients as a source of pathogenic forms of TDP-43, and healthy human cells (lymphoblasts, myoblasts, neuroblastoma SH-SY5Y, or osteosarcoma U2OS) as recipient cells to investigate the seeding and spread of TDP-43 proteinopathy. Furthermore, we evaluated the potential of targeting TDP-43 phosphorylation with a CK-1 inhibitor to prevent the propagation of the pathology. The results presented herein indicate that pathogenic forms of TDP-43 are secreted into the extracellular medium of sporadic ALS lymphoblasts and could be transported by extracellular vesicles, spreading TDP-43 pathology to healthy cells. Moreover, tunneling nanotubes have also been discovered in pathological cells and may be involved in the transport of TDP-43. Interestingly, targeting TDP-43 phosphorylation with an in-house designed CK-1 inhibitor (IGS2.7) was sufficient to halt TDP-43 pathology transmission, in addition to its known effects on restoring the homeostasis of TDP-43 protein in patients-derived cells.
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Esclerose Lateral Amiotrófica , Neuroblastoma , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Caseína Quinase I , Proteínas de Ligação a DNA/metabolismoRESUMO
Cell division cycle 7 kinase (CDC7) has been found overexpressed in many cancer cell lines being also one of the kinases involved in the nuclear protein TDP-43 phosphorylation in vivo. Thus, inhibitors of CDC7 are emerging drug candidates for the treatment of oncological and neurodegenerative unmet diseases. All the known CDC7 inhibitors are ATP-competitives, lacking of selectivity enough for success in clinical trials. As allosteric sites are less conserved among kinase proteins, discovery of allosteric modulators of CDC7 is a great challenge and opportunity in this field.Using different computational approaches, we have here identified new druggable cavities on the human CDC7 structure and subsequently selective CDC7 inhibitors with allosteric modulation mainly targeting the pockets where the interaction between this kinase and its activator DBF4 takes place.
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Proteínas Nucleares , Proteínas Serina-Treonina Quinases , Humanos , Fosforilação , Sítio Alostérico , Linhagem Celular , Ciclo Celular , Proteínas de Ciclo CelularRESUMO
Saffron has many pharmacological properties in addition to being a frequently used food seasoning. Crocin and picrocrocin which accumulate in saffron stigma, are responsible for these pharmacological properties. These natural products have health-promoting effects for the prevention and treatment of numerous diseases, including age-related cognitive and memory disfunction. Currently, crocin and picrocrocin are obtained from saffron, considered as the spice with the highest price in the market. To develop an efficient and low-cost approach to producing these compounds with high bioactivity, biosynthetic genes isolated from saffron can be exploited in the metabolic engineering of heterologous hosts and the production of crocins in productive crop plants. Recently, we engineered tomato fruit producing crocins (Tomafran). In this study, we demonstrated that crocin-rich extract, encapsulated in chitosan or in exosomes may function as a neuroprotective strategy. Crocins contained in the Tomafran extracts and much lower doses in chitosan nanoparticles or exosomes were enough to rescue the neuroblastoma cell line SH-SY5Y after damage caused by okadaic acid. Our results confirm the neuroprotective effect of Tomafran and its exosomes that may be useful for the delay or prevention of neurodegenerative disorders such as Alzheimer's disease.
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Advances in neonatology have significantly reduced mortality rates due to prematurity. However, complications of prematurity have barely changed in recent decades. Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most severe complications of prematurity, and these children are prone to suffer short- and long-term sequelae, including cerebral palsy, cognitive and motor impairments, or neuropsychiatric disorders. Nevertheless, GM-IVH has no successful treatment. VP3.15 is a small, heterocyclic molecule of the 5-imino-1,2,4-thiadiazole family with a dual action as a phosphodiesterase 7 and glycogen synthase kinase-3ß (GSK-3ß) inhibitor. VP3.15 reduces neuroinflammation and neuronal loss in other neurodegenerative disorders and might ameliorate complications associated with GM-IVH. We administered VP3.15 to a mouse model of GM-IVH. VP3.15 reduces the presence of hemorrhages and microglia in the short (P14) and long (P110) term. It ameliorates brain atrophy and ventricle enlargement while limiting tau hyperphosphorylation and neuronal and myelin basic protein loss. VP3.15 also improves proliferation and neurogenesis as well as cognition after the insult. Interestingly, plasma gelsolin levels, a feasible biomarker of brain damage, improved after VP3.15 treatment. Altogether, our data support the beneficial effects of VP3.15 in GM-IVH by ameliorating brain neuroinflammatory, vascular and white matter damage, ultimately improving cognitive impairment associated with GM-IVH.
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INTRODUCTION: The clinical effect of domperidone against COVID-19 has been investigated in a double-blind phase III clinical trial (EudraCT number 2021-001228-17). Domperidone has shown in vitro antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential immudolatory properties through the stimulation of prolactin secretion. PATIENTS AND METHODS: The efficacy of oral domperidone plus standard of care (SOC; n = 87) versus placebo plus SOC (n = 86) was evaluated in a 28-day randomized double-blind multicentre study in primary health care centres. A total of 173 outpatients with mild-to-moderate COVID-19 were included. Three daily doses of 10 mg (30 mg/day) of domperidone or placebo were administered for 7 days. Reduction of viral load on day 4 was the primary efficay endpoint. It was estimated in saliva samples by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), as the cycle thresholds detected ORF1ab, N Protein and S Protein genes. RESULTS: A significant reduction in the viral load was observed (p < 0.001) from baseline to days 4, 7 and 14 of the three genes studied with non-significant differences between domperidone and placebo groups. Twenty-three patients (13.3%) experienced adverse events, 14 patients in the domperidone group (16.1%) and 9 patients in the placebo group (10.5%). No patients needed to be hospitalized. CONCLUSION: Results do not prove the use of domperidone as antiviral in patients with COVID-19.
A 28-day double-blind clinical trial was performed to investigate the antiviral effect of domperidone, 30 mg/day for 7 days (n = 87) versus placebo (n = 86) in outpatients with mild-to-moderate COVID-19.The primary efficacy endpoint was the reduction of viral load on day 4 as compared with baseline, estimated as the cycle thresholds to detect ORF1ab, N Protein and S Protein genes by RT-qPCR in saliva samples.The study findings do not prove the use of domperidone as antiviral in patients with COVID-19.
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COVID-19 , Humanos , SARS-CoV-2 , Domperidona/uso terapêutico , Método Duplo-Cego , Carga Viral , Resultado do Tratamento , Antivirais/uso terapêutico , Atenção Primária à SaúdeRESUMO
3D bioprinting is revolutionizing the fields of personalized and precision medicine by enabling the manufacturing of bioartificial implants that recapitulate the structural and functional characteristics of native tissues. However, the lack of quantitative and noninvasive techniques to longitudinally track the function of implants has hampered clinical applications of bioprinted scaffolds. In this study, multimaterial 3D bioprinting, engineered nanoparticles (NPs), and spectral photon-counting computed tomography (PCCT) technologies are integrated for the aim of developing a new precision medicine approach to custom-engineer scaffolds with traceability. Multiple CT-visible hydrogel-based bioinks, containing distinct molecular (iodine and gadolinium) and NP (iodine-loaded liposome, gold, methacrylated gold (AuMA), and Gd2 O3 ) contrast agents, are used to bioprint scaffolds with varying geometries at adequate fidelity levels. In vitro release studies, together with printing fidelity, mechanical, and biocompatibility tests identified AuMA and Gd2 O3 NPs as optimal reagents to track bioprinted constructs. Spectral PCCT imaging of scaffolds in vitro and subcutaneous implants in mice enabled noninvasive material discrimination and contrast agent quantification. Together, these results establish a novel theranostic platform with high precision, tunability, throughput, and reproducibility and open new prospects for a broad range of applications in the field of precision and personalized regenerative medicine.
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Bioimpressão , Iodo , Camundongos , Animais , Bioimpressão/métodos , Reprodutibilidade dos Testes , Engenharia Tecidual/métodos , Tomografia Computadorizada por Raios X , Impressão Tridimensional , Alicerces Teciduais/químicaRESUMO
Chemical libraries have become of utmost importance to boost drug discovery processes. It is widely accepted that the quality of a chemical library depends, among others, on its availability and chemical diversity which help in rising the chances of finding good hits. In this regard, our group has developed a source for useful chemicals named Medicinal and Biological Chemistry (MBC) library. It originates from more than 30 years of experience in drug design and discovery of our research group and has successfully provided effective hits for neurological, neurodegenerative and infectious diseases. Moreover, in the last years, the European research infrastructure for chemical biology EU-OPENSCREEN has generated the European Chemical Biology library (ECBL) to be used as a source of hits for drug discovery. Here we present and discuss the updated version of the MBC library (MBC v.2022), enriched with new scaffolds and containing more than 2,500 compounds together with ECBL that collects about 100,000 small molecules. To properly address the improved potentialities of the new version of our MBC library in drug discovery, up to 44 among physicochemical and pharmaceutical properties have been calculated and compared with those of other well-known publicly available libraries. For comparison, we have used ZINC20, DrugBank, ChEMBL library, ECBL and NuBBE along with an approved drug library. Final results allowed to confirm the competitive chemical space covered by MBC v.2022 and ECBL together with suitable drug-like properties. In all, we can affirm that these two libraries represent an interesting source of new hits for drug discovery.
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Introduction: TDP-43 proteinopathy in Alzheimer's disease (AD) patients is recently emerging as a relevant pathomolecular event that may have been overlooked. Recent results in immortalized lymphocytes from AD patients have shown not only an increase of post-translational modifications in TDP-43, such as hyperphosphorylation and fragmentation, but also its prionic behaviour and cell-to-cell disease transmission. With the main goal to advance therapeutic interventions, we present in this work different kinase inhibitors with potential to restore this pathological mechanism. Methodology: We have used immortalized lymphocytes from healthy controls and AD severe patients to evaluate the correction of TDP-43 pathology after the treatment with previously synthetized TTBK1 and CK1 inhibitors. Moreover we used the conditioned mediums of these cells to perform different disease propagation experiments. Results: TDP-43 pathology observed in lymphoblasts from severe AD patients is reduced after the treatment with TTBK1 and CK1 inhibitors (decreasing phosphorylation and increasing nuclear localisation), Furthermore, the significant increase in TDP-43 phosphorylation, cytoplasmic accumulation and aberrant F-actin protrusions (TNT-like structures) observed in control cells growing in CM from AD lymphoblasts were abolished when the CM from AD lymphoblasts treated with previously reported TTBK1 and CK1 inhibitors were used. In addition, the cytosolic transport mediated by molecular motors of the receptor cells was altered with the induced TDP-43 pathology, but it was not produced with the abovementioned pretreated CMs. Conclusion: TTBK1 and CK1 inhibitors, specially VNG1.47 and IGS2.7 compounds, restore TDP-43 pathology and avoid cell-to-cell propagation in immortalized lymphocytes from AD patients, being excellent candidates for the future therapy of this prevalent and devastating disease.
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Selective and brain-permeable protein kinase inhibitors are in preclinical development for treating neurodegenerative diseases. Among them, MLK3 inhibitors, with a potent neuroprotective biological action have emerged as valuable agents for the treatment of pathologies such as Alzheimer's, Parkinson's disease and amyotrophic lateral sclerosis. In fact, one MLK3 inhibitor, CEP-1347, reached clinical trials for Parkinson's disease. Additionally, another compound called prostetin/12k, a potent and rather selective MLK3 inhibitor has started clinical development for ALS based on its motor neuron protection in both in vitro and in vivo models. In this review, we will focus on the role of MLK3 in neuron-related cell death processes, neurodegenerative diseases, and the potential advantages of targeting this kinase through pharmacological modulation for neuroprotective treatment.
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Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , MAP Quinase Quinase Quinases , Morte Celular , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Adhesive tissue engineering scaffolds (ATESs) have emerged as an innovative alternative means, replacing sutures and bioglues, to secure the implants onto target tissues. Relying on their intrinsic tissue adhesion characteristics, ATES systems enable minimally invasive delivery of various scaffolds. This study investigates development of the first class of 3D bioprinted ATES constructs using functionalized hydrogel bioinks. Two ATES delivery strategies, in situ printing onto the adherend versus printing and then transferring to the target surface, are tested using two bioprinting methods, embedded versus air printing. Dopamine-modified methacrylated hyaluronic acid (HAMA-Dopa) and gelatin methacrylate (GelMA) are used as the main bioink components, enabling fabrication of scaffolds with enhanced adhesion and crosslinking properties. Results demonstrate that dopamine modification improved adhesive properties of the HAMA-Dopa/GelMA constructs under various loading conditions, while maintaining their structural fidelity, stability, mechanical properties, and biocompatibility. While directly printing onto the adherend yields superior adhesive strength, embedded printing followed by transfer to the target tissue demonstrates greater potential for translational applications. Together, these results demonstrate the potential of bioprinted ATESs as off-the-shelf medical devices for diverse biomedical applications.
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Bioimpressão , Adesivos Teciduais , Engenharia Tecidual/métodos , Hidrogéis/química , Adesivos , Bioimpressão/métodos , Dopamina , Gelatina/química , Metacrilatos/química , Impressão TridimensionalRESUMO
Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.
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Ebolavirus , Doença pelo Vírus Ebola , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Replicação ViralRESUMO
Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.
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Inibidores da Fosfodiesterase 4 , Esquistossomose , Animais , Humanos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Schistosoma mansoni , Benzamidas/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Esquistossomose/tratamento farmacológico , Nucleotídeos CíclicosRESUMO
Fishery residues are abundant raw materials that also provide numerous metabolites with high added value. Their classic valorization includes energy recovery, composting, animal feed, and direct deposits in landfills or oceans along with the environmental impacts that this entails. However, through extraction processes, they can be transformed into new compounds with high added value, offering a more sustainable solution. The aim of this study was to optimize the extraction process of chitosan and fish gelatin from fishery waste and their revalorization as active biopolymers. We successfully optimized the chitosan extraction process, achieving a yield of 20.45% and a deacetylation degree of 69.25%. For the fish gelatin extraction process, yields of 11.82% for the skin and 2.31% for the bone residues were achieved. In addition, it was demonstrated that simple purification steps using activated carbon improve the gelatin's quality significantly. Finally, biopolymers based on fish gelatin and chitosan showed excellent bactericidal capabilities against Escherichia coli and Listeria innocua. For this reason, these active biopolymers can stop or decrease bacterial growth in their potential food packaging applications. In view of the low technological transfer and the lack of information about the revalorization of fishery waste, this work offers extraction conditions with good yields that can be easily implemented in the existing industrial fabric, reducing costs and supporting the economic development of the fish processing sector and the creation of value from its waste.
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BACKGROUND: Despite being a new entity, there is a large amount of information on the characteristics of SARS-CoV-2 infection and the symptoms of the acute phase; however, there are still many unknowns about the clinical features and pathophysiology of post-COVID syndrome. Refractory chronic cough is one of the most prevalent symptoms and carries both a medical problem and a social stigma. Many recent studies have highlighted the role of SARS-CoV-2 neurotropism, but no studies have demonstrated vagus nerve neuropathy as a cause of persistent chronic cough or other COVID-19 long-term effects. OBJECTIVE: The main objective was to assess the involvement of the vagus nerve neuropathy as a cause of chronic cough and other post-COVID syndrome symptoms. MATERIAL AND METHODS: This was a single-center observational study with prospective clinical data collected from 38 patients with chronic cough and post-COVID-19 syndrome. Clinical characteristics and laryngeal electromyographic findings were analyzed. RESULTS: Clinical data from 38 patients with chronic cough after 12 weeks of the acute phase of COVID-19 infection were analyzed. Of these patients, 81.6% suffered from other post-COVID conditions and, 73.6% reported fluctuating evolution of symptoms. Laryngeal electromyography (LEMG) of the thyroarytenoid (TA) muscles and cricothyroid (CT) muscles was pathological in 76.3% of the patients. Of the patients with abnormal LEMG, chronic denervation was the most frequent finding (82.8%), 10.3% presented acute denervation signs, and 6.9% presented myopathic pattern in LEMG. CONCLUSIONS: LEMG studies suggest the existence of postviral vagus nerve neuropathy after SARS-CoV-2 infection that could explain chronic cough in post-COVID syndrome.
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COVID-19 , Doenças do Sistema Nervoso Periférico , Humanos , Eletromiografia , Tosse , Estudos Prospectivos , Síndrome de COVID-19 Pós-Aguda , COVID-19/complicações , SARS-CoV-2 , Nervo Vago , Músculos Laríngeos , Doença CrônicaRESUMO
BACKGROUND AND AIM: Although sensorineural hearing loss may have different aetiologies, we focused on autoimmune hearing loss since it may be reversible with corticosteroid therapy; this entity is sometimes associated with systemic autoimmune diseases. Hashimoto's thyroiditis or chronic autoimmune thyroiditis shows antibodies and may be harmful to hearing thresholds regardless of hypothyroidism effect. To date this effect has not been sufficiently studied and never with extended high frequencies. The aim of this work is to study by age groups whether hearing thresholds in the human auditory range (128-20,000Hz) are affected in Hashimoto's disease. MATERIALS AND METHODS: Two groups of 128 patients affected by Hashimoto's thyroiditis were included. First group: patients with pathological antithyroid antibodies who do not need L-thyroxine treatment. Second group: patients controlled with L-thyroxine substitutive treatment. Audiometric threshold study comparing between the groups of patients and a group of 209 controls was performed. All patients underwent complete otorhinolaryngological examination, antithyroid antibodies, TSH, T3 and T4 blood levels, tympanometry, conventional pure-tone audiometry, and extended-high-frequency audiometry. RESULTS: All patients were women. Both groups showed worst audiometric thresholds than the control group; both study groups showed worse hearing than controls, this difference was statistically significant in all frequencies. In the 8-20kHz frequency range, this difference was more than 10dB, and in the 9-16kHz and 20kHz range this difference was more than 20dB. When separated by age groups, in younger subjects (20-29 years) these differences were found in all frequencies, except for conversational frequencies (500-4,000Hz); between 30 and 49 years the difference is statistically significant in all frequencies; and from 50 to 69 years differences are found, especially in the conversational frequencies. CONCLUSIONS: This first work studying the human auditory range in the chronic autoimmune thyroiditis or Hashimoto's thyroiditis confirms that hearing loss related to the autoimmune disorder predominates at extended-high-frequencies initially. But ends up involving all frequencies in pure-tone conventional audiometry, then it may be detected in routine clinical tests. These results support the role of extended-high-frequencies audiometry to diagnose subclinical hearing loss in patients affected by Hashimoto's thyroiditis.
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Doença de Hashimoto , Perda Auditiva , Tireoidite Autoimune , Humanos , Feminino , Adulto Jovem , Adulto , Masculino , Tireoidite Autoimune/complicações , Doença de Hashimoto/complicações , Tiroxina/uso terapêutico , Perda Auditiva/complicações , Audiometria de Tons PurosRESUMO
BACKGROUND: PDEs regulate cAMP levels which is critical for PKA activity-dependent activation of CREB-mediated transcription in learning and memory. Inhibitors of PDEs like PDE4 and Pde7 improve learning and memory in rodents. However, the role of PDE7 in cognition or learning and memory has not been reported yet. METHODS: Therefore, we aimed to explore the cognitive effects of a PDE7 subtype, PDE7a, using combined pharmacological and genetic approaches. RESULTS: PDE7a-nko mice showed deficient working memory, impaired novel object recognition, deficient spatial learning & memory, and contextual fear memory, contrary to enhanced cued fear memory, highlighting the potential opposite role of PDE7a in the hippocampal neurons. Further, pharmacological inhibition of PDE7 by AGF2.20 selectively strengthens cued fear memory in C57BL/6 J mice, decreasing its extinction but did not affect cognitive processes assessed in other behavioral tests. The further biochemical analysis detected deficient cAMP in neural cell culture with genetic excision of the PDE7a gene, as well as in the hippocampus of PDE7a-nko mice in vivo. Importantly, we found overexpression of PKA-R and the reduced level of pPKA-C in the hippocampus of PDE7a-nko mice, suggesting a novel mechanism of the cAMP regulation by PDE7a. Consequently, the decreased phosphorylation of CREB, CAMKII, eif2a, ERK, and AMPK, and reduced total level of NR2A have been found in the brain of PDE7a-nko animals. Notably, genetic excision of PDE7a in neurons was not able to change the expression of NR2B, BDNF, synapsin1, synaptophysin, or snap25. CONCLUSION: Altogether, our current findings demonstrated, for the first time, the role of PDE7a in cognitive processes. Future studies will untangle PDE7a-dependent neurobiological and molecular-cellular mechanisms related to cAMP-associated disorders.
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Memória de Curto Prazo , Aprendizagem Espacial , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Medo , Hipocampo/metabolismo , Camundongos Endogâmicos C57BL , Sinaptofisina/metabolismo , Memória , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismoRESUMO
Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.
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[This corrects the article DOI: 10.1371/journal.ppat.1010631.].