Publications by authors named "Nélio Gonçalves"

18 Publications

  • Page 1 of 1

In Vitro and In Vivo Tumor Models for the Evaluation of Anticancer Nanoparticles.

Adv Exp Med Biol 2021 ;1295:271-299

CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, Coimbra, Portugal.

Multiple studies about tumor biology have revealed the determinant role of the tumor microenvironment in cancer progression, resulting from the dynamic interactions between tumor cells and surrounding stromal cells within the extracellular matrix. This malignant microenvironment highly impacts the efficacy of anticancer nanoparticles by displaying drug resistance mechanisms, as well as intrinsic physical and biochemical barriers, which hamper their intratumoral accumulation and biological activity.Currently, two-dimensional cell cultures are used as the initial screening method in vitro for testing cytotoxic nanocarriers. However, this fails to mimic the tumor heterogeneity, as well as the three-dimensional tumor architecture and pathophysiological barriers, leading to an inaccurate pharmacological evaluation.Biomimetic 3D in vitro tumor models, on the other hand, are emerging as promising tools for more accurately assessing nanoparticle activity, owing to their ability to recapitulate certain features of the tumor microenvironment and thus provide mechanistic insights into nanocarrier intratumoral penetration and diffusion rates.Notwithstanding, in vivo validation of nanomedicines remains irreplaceable at the preclinical stage, and a vast variety of more advanced in vivo tumor models is currently available. Such complex animal models (e.g., genetically engineered mice and patient-derived xenografts) are capable of better predicting nanocarrier clinical efficiency, as they closely resemble the heterogeneity of the human tumor microenvironment.Herein, the development of physiologically more relevant in vitro and in vivo tumor models for the preclinical evaluation of anticancer nanoparticles will be discussed, as well as the current limitations and future challenges in clinical translation.
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http://dx.doi.org/10.1007/978-3-030-58174-9_12DOI Listing
February 2021

Cancer Stem Cells and Nucleolin as Drivers of Carcinogenesis.

Pharmaceuticals (Basel) 2021 Jan 13;14(1). Epub 2021 Jan 13.

CNC-Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), Faculty of Medicine (Polo 1), University of Coimbra, Rua Larga, 3004-504 Coimbra, Portugal.

Cancer, one of the most mortal diseases worldwide, is characterized by the gain of specific features and cellular heterogeneity. Clonal evolution is an established theory to explain heterogeneity, but the discovery of cancer stem cells expanded the concept to include the hierarchical growth and plasticity of cancer cells. The activation of epithelial-to-mesenchymal transition and its molecular players are widely correlated with the presence of cancer stem cells in tumors. Moreover, the acquisition of certain oncological features may be partially attributed to alterations in the levels, location or function of nucleolin, a multifunctional protein involved in several cellular processes. This review aims at integrating the established hallmarks of cancer with the plasticity of cancer cells as an emerging hallmark; responsible for tumor heterogeneity; therapy resistance and relapse. The discussion will contextualize the involvement of nucleolin in the establishment of cancer hallmarks and its application as a marker protein for targeted anticancer therapies.
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http://dx.doi.org/10.3390/ph14010060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828541PMC
January 2021

Current challenges and emerging opportunities of CAR-T cell therapies.

J Control Release 2020 03 30;319:246-261. Epub 2019 Dec 30.

CNC - Center for Neurosciences and Cell Biology, University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, 3004-504 Coimbra, Portugal; FFUC - Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address:

Infusion of chimeric antigen receptor (CAR)-genetically modified T cells (CAR-T cells) have led to remarkable clinical responses and cancer remission in patients suffering from relapsed or refractory B-cell malignancies. This is a new form of adoptive T cell therapy (ACT), whereby the artificial CAR enables the redirection of T cells endogenous antitumor activity towards a predefined tumor-associated antigen, leading to the elimination of a specific tumor. The early success in blood cancers has prompted the US Food and Drug Administration (FDA) to approve the first CAR-T cell therapies for the treatment of CD19-positive leukemias and lymphomas in 2017. Despite the emergence of CAR-T cells as one of the latest breakthroughs of cancer immunotherapies, their wider application has been hampered by specific life-threatening toxicities, and a substantial lack of efficacy in the treatment of solid tumors, owing to the strong immunosuppressive tumor microenvironment and the paucity of reliable tumor-specific targets. Herein, besides providing an overview of the emerging CAR-technologies and current clinical applications, the major hurdles of CAR-T cell therapies will be discussed, namely treatment-related life-threatening toxicities and the obstacles posed by the immunosupressive tumor-microenvironment of solid tumors, as well as the next-generation strategies currently designed to simultaneously improve safety and efficacy of CAR-T cell therapies in vivo.
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http://dx.doi.org/10.1016/j.jconrel.2019.12.047DOI Listing
March 2020

Synaptic and memory dysfunction in a β-amyloid model of early Alzheimer's disease depends on increased formation of ATP-derived extracellular adenosine.

Neurobiol Dis 2019 12 5;132:104570. Epub 2019 Aug 5.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; FMUC-Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal. Electronic address:

Adenosine A receptors (AR) overfunction causes synaptic and memory dysfunction in early Alzheimer's disease (AD). In a β-amyloid (Aβ)-based model of early AD, we now unraveled that this involves an increased synaptic release of ATP coupled to an increased density and activity of ecto-5'-nucleotidase (CD73)-mediated formation of adenosine selectively activating AR. Thus, CD73 inhibition with α,β-methylene-ADP impaired long-term potentiation (LTP) in mouse hippocampal slices, which is occluded upon previous superfusion with the AR antagonist SCH58261. Furthermore, α,β-methylene-ADP did not alter LTP amplitude in global AR knockout (KO) and in forebrain neuron-selective AR-KO mice, but inhibited LTP amplitude in astrocyte-selective AR-KO mice; this shows that CD73-derived adenosine solely acts on neuronal AR. In agreement with the concept that ATP is a danger signal in the brain, ATP release from nerve terminals is increased after intracerebroventricular Aβ administration, together with CD73 and AR upregulation in hippocampal synapses. Importantly, this increased CD73 activity is critically required for Aβ to impair synaptic plasticity and memory since Aβ-induced synaptic and memory deficits were eliminated in CD73-KO mice. These observations establish a key regulatory role of CD73 activity over neuronal AR and imply CD73 as a novel target for modulation of early AD.
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http://dx.doi.org/10.1016/j.nbd.2019.104570DOI Listing
December 2019

Neuronal Adenosine A2A Receptors Are Critical Mediators of Neurodegeneration Triggered by Convulsions.

eNeuro 2018 Nov-Dec;5(6). Epub 2018 Dec 26.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.

Neurodegeneration is a process transversal to neuropsychiatric diseases and the understanding of its mechanisms should allow devising strategies to prevent this irreversible step in brain diseases. Neurodegeneration caused by seizures is a critical step in the aggravation of temporal lobe epilepsy, but its mechanisms remain undetermined. Convulsions trigger an elevation of extracellular adenosine and upregulate adenosine A receptors (AR), which have been associated with the control of neurodegenerative diseases. Using the rat and mouse kainate model of temporal lobe epilepsy, we now tested whether AR control convulsions-induced hippocampal neurodegeneration. The pharmacological or genetic blockade of AR did not affect kainate-induced convulsions but dampened the subsequent neurotoxicity. This neurotoxicity began with a rapid AR upregulation within glutamatergic synapses (within 2 h), through local translation of synaptic AR mRNA. This bolstered AR-mediated facilitation of glutamate release and of long-term potentiation (LTP) in CA1 synapses (4 h), triggered a subsequent synaptotoxicity, heralded by decreased synaptic plasticity and loss of synaptic markers coupled to calpain activation (12 h), that predated overt neuronal loss (24 h). All modifications were prevented by the deletion of AR selectively in forebrain neurons. This shows that synaptic AR critically control synaptic excitotoxicity, which underlies the development of convulsions-induced neurodegeneration.
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http://dx.doi.org/10.1523/ENEURO.0385-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325550PMC
March 2019

Adenosine A Receptors in the Rat Prelimbic Medial Prefrontal Cortex Control Delay-Based Cost-Benefit Decision Making.

Front Mol Neurosci 2018 20;11:475. Epub 2018 Dec 20.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Adenosine A receptors (ARs) were recently described to control synaptic plasticity and network activity in the prefrontal cortex (PFC). We now probed the role of these PFC AR by evaluating the behavioral performance (locomotor activity, anxiety-related behavior, cost-benefit decision making and working memory) of rats upon downregulation of AR selectively in the prelimbic medial PFC (PLmPFC) via viral small hairpin RNA targeting the AR (shAR). The most evident alteration observed in shAR-treated rats, when compared to sh-control (shCTRL)-treated rats, was a decrease in the choice of the large reward upon an imposed delay of 15 s assessed in a T-maze-based cost-benefit decision-making paradigm, suggestive of impulsive decision making. Spontaneous locomotion in the open field was not altered, suggesting no changes in exploratory behavior. Furthermore, rats treated with shAR in the PLmPFC also displayed a tendency for higher anxiety levels in the elevated plus maze (less entries in the open arms), but not in the open field test (time spent in the center was not affected). Finally, working memory performance was not significantly altered, as revealed by the spontaneous alternation in the Y-maze test and the latency to reach the platform in the repeated trial Morris water maze. These findings constitute the first direct demonstration of a role of PFC AR in the control of behavior in physiological conditions, showing their major contribution for the control of delay-based cost-benefit decisions.
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http://dx.doi.org/10.3389/fnmol.2018.00475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306464PMC
December 2018

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.

Hum Mol Genet 2018 10;27(20):3528-3541

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.
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http://dx.doi.org/10.1093/hmg/ddy264DOI Listing
October 2018

Astrocytic A2A receptors: Novel targets to manage brain disorders: .

Porto Biomed J 2017 Sep-Oct;2(5):178-179. Epub 2017 Sep 1.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.

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http://dx.doi.org/10.1016/j.pbj.2017.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6806793PMC
September 2017

Caffeine alleviates progressive motor deficits in a transgenic mouse model of spinocerebellar ataxia.

Ann Neurol 2017 Mar 17;81(3):407-418. Epub 2017 Feb 17.

Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Objective: Machado-Joseph disease (MJD) is a neurodegenerative spinocerebellar ataxia (SCA) associated with an expanded polyglutamine tract within ataxin-3 for which there is currently no available therapy. We previously showed that caffeine, a nonselective adenosine receptor antagonist, delays the appearance of striatal damage resulting from expression of full-length mutant ataxin-3. Here we investigated the ability of caffeine to alleviate behavioral deficits and cerebellar neuropathology in transgenic mice with a severe ataxia resulting from expression of a truncated fragment of polyglutamine-expanded ataxin-3 in Purkinje cells.

Methods: Control and transgenic c57Bl6 mice expressing in the mouse cerebella a truncated form of human ataxin-3 with 69 glutamine repeats were allowed to freely drink water or caffeinated water (1g/L). Treatments began at 7 weeks of age, when motor and ataxic phenotype emerges in MJD mice, and lasted up to 20 weeks. Mice were tested in a panel of locomotor behavioral paradigms, namely rotarod, beam balance and walking, pole, and water maze cued-platform version tests, and then sacrificed for cerebellar histology.

Results: Caffeine consumption attenuated the progressive loss of general and fine-tuned motor function, balance, and grip strength, in parallel with preservation of cerebellar morphology through decreasing the loss of Purkinje neurons and the thinning of the molecular layer in different folia. Caffeine also rescued the putative striatal-dependent executive and cognitive deficiencies in MJD mice.

Interpretation: Our findings provide the first in vivo demonstration that caffeine intake alleviates behavioral disabilities in a severely impaired animal model of SCA. Ann Neurol 2017;81:407-418.
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http://dx.doi.org/10.1002/ana.24867DOI Listing
March 2017

Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors.

Nat Commun 2016 06 17;7:11915. Epub 2016 Jun 17.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, CNRS UMR 5297, F-33000 Bordeaux, France.

Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.
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http://dx.doi.org/10.1038/ncomms11915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915032PMC
June 2016

Adenosine A Receptors in the Amygdala Control Synaptic Plasticity and Contextual Fear Memory.

Neuropsychopharmacology 2016 11 17;41(12):2862-2871. Epub 2016 Jun 17.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

The consumption of caffeine modulates working and reference memory through the antagonism of adenosine A receptors (ARs) controlling synaptic plasticity processes in hippocampal excitatory synapses. Fear memory essentially involves plastic changes in amygdala circuits. However, it is unknown if ARs in the amygdala regulate synaptic plasticity and fear memory. We report that ARs in the amygdala are enriched in synapses and located to glutamatergic synapses, where they selectively control synaptic plasticity rather than synaptic transmission at a major afferent pathway to the amygdala. Notably, the downregulation of ARs selectively in the basolateral complex of the amygdala, using a lentivirus with a silencing shRNA (small hairpin RNA targeting AR (shAR)), impaired fear acquisition as well as Pavlovian fear retrieval. This is probably associated with the upregulation and gain of function of ARs in the amygdala after fear acquisition. The importance of ARs to control fear memory was further confirmed by the ability of SCH58261 (0.1 mg/kg; AR antagonist), caffeine (5 mg/kg), but not DPCPX (0.5 mg/kg; AR antagonist), treatment for 7 days before fear conditioning onwards, to attenuate the retrieval of context fear after 24-48 h and after 7-8 days. These results demonstrate that amygdala ARs control fear memory and the underlying process of synaptic plasticity in this brain region. This provides a neurophysiological basis for the association between AR polymorphisms and phobia or panic attacks in humans and prompts a therapeutic interest in ARs to manage fear-related pathologies.
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http://dx.doi.org/10.1038/npp.2016.98DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061896PMC
November 2016

Depression as a Glial-Based Synaptic Dysfunction.

Front Cell Neurosci 2015 22;9:521. Epub 2016 Jan 22.

CNC - Center for Neuroscience and Cell Biology, University of CoimbraCoimbra, Portugal; Faculty of Medicine, University of CoimbraCoimbra, Portugal.

Recent studies combining pharmacological, behavioral, electrophysiological and molecular approaches indicate that depression results from maladaptive neuroplastic processes occurring in defined frontolimbic circuits responsible for emotional processing such as the prefrontal cortex, hippocampus, amygdala and ventral striatum. However, the exact mechanisms controlling synaptic plasticity that are disrupted to trigger depressive conditions have not been elucidated. Since glial cells (astrocytes and microglia) tightly and dynamically interact with synapses, engaging a bi-directional communication critical for the processing of synaptic information, we now revisit the role of glial cells in the etiology of depression focusing on a dysfunction of the "quad-partite" synapse. This interest is supported by the observations that depressive-like conditions are associated with a decreased density and hypofunction of astrocytes and with an increased microglia "activation" in frontolimbic regions, which is expected to contribute for the synaptic dysfunction present in depression. Furthermore, the traditional culprits of depression (glucocorticoids, biogenic amines, brain-derived neurotrophic factor, BDNF) affect glia functioning, whereas antidepressant treatments (serotonin-selective reuptake inhibitors, SSRIs, electroshocks, deep brain stimulation) recover glia functioning. In this context of a quad-partite synapse, systems modulating glia-synapse bidirectional communication-such as the purinergic neuromodulation system operated by adenosine 5'-triphosphate (ATP) and adenosine-emerge as promising candidates to "re-normalize" synaptic function by combining direct synaptic effects with an ability to also control astrocyte and microglia function. This proposed triple action of purines to control aberrant synaptic function illustrates the rationale to consider the interference with glia dysfunction as a mechanism of action driving the design of future pharmacological tools to manage depression.
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http://dx.doi.org/10.3389/fncel.2015.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722129PMC
February 2016

Neuropeptide Y mitigates neuropathology and motor deficits in mouse models of Machado-Joseph disease.

Hum Mol Genet 2015 Oct 27;24(19):5451-63. Epub 2015 Jul 27.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-517, Portugal, Faculty of Pharmacy, University of Coimbra, Coimbra 3000-548, Portugal,

Machado-Joseph disease (MJD) is a fatal, dominantly inherited neurodegenerative disorder associated with an expanded polyglutamine tract within the ataxin-3 protein, and characterized by progressive impairment of motor coordination, associated with neurodegeneration of specific brain regions, including cerebellum and striatum. The currently available therapies do not allow modification of disease progression. Neuropeptide Y (NPY) has been shown to exert potent neuroprotective effects by multiple pathways associated with the MJD mechanisms of disease. Thus, we evaluated NPY levels in MJD and investigated whether raising NPY by gene transfer would alleviate neuropathological and behavioural deficits in cerebellar and striatal mouse models of the disease. For that, a cerebellar transgenic and a striatal lentiviral-based models of MJD were used. NPY overexpression in the affected brain regions in these two mouse models was obtained by stereotaxic injection of adeno-associated viral vectors encoding NPY. Up to 8 weeks after viral injection, balance and motor coordination behaviour and neuropathology were analysed. We observed that NPY levels were decreased in two MJD patients' cerebella and in striata and cerebella of disease mouse models. Furthermore, overexpression of NPY alleviated the motor coordination impairments and attenuated the related neuropathological parameters, preserving cerebellar volume and granular layer thickness, reducing striatal lesion and decreasing mutant ataxin-3 aggregation. Additionally, NPY mediated increase of brain-derived neurotrophic factor levels and decreased neuroinflammation markers. Our data suggest that NPY is a potential therapeutic strategy for MJD.
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http://dx.doi.org/10.1093/hmg/ddv271DOI Listing
October 2015

Calpain inhibition reduces ataxin-3 cleavage alleviating neuropathology and motor impairments in mouse models of Machado-Joseph disease.

Hum Mol Genet 2014 Sep 9;23(18):4932-44. Epub 2014 May 9.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-517, Portugal Faculty of Pharmacy, University of Coimbra, Coimbra 3000-548, Portugal

Machado-Joseph Disease (MJD) is the most prevalent autosomal dominantly inherited cerebellar ataxia. It is caused by an expanded CAG repeat in the ATXN3 gene, which translates into a polyglutamine tract within the ataxin-3 protein. Present treatments are symptomatic and do not prevent disease progression. As calpain overactivation has been shown to contribute to mutant ataxin-3 proteolysis, translocation to the nucleus, inclusions formation and neurodegeneration, we investigated the potential role of calpain inhibition as a therapeutic strategy to alleviate MJD pathology. For this purpose, we administered orally the calpain inhibitor BDA-410 to a lentiviral mouse model of MJD. Western-blot and immunohistochemical analysis revealed the presence of N- and C-terminal mutant ataxin-3 fragments and the colocalization of large inclusions with cleaved caspase-3 in the mice brain. Oral administration of the calpain inhibitor BDA-410 decreased both fragments formation and full-length ataxin-3 levels, reduced aggregation of mutant ataxin-3 and prevented cell injury and striatal and cerebellar degeneration. Importantly, in correlation with the preserved cerebellar morphology, BDA-410 prevented motor behavioural deficits. In conclusion, BDA-410 alleviates Machado-Joseph neuropathology and may therefore be an effective therapeutic option for MJD.
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http://dx.doi.org/10.1093/hmg/ddu209DOI Listing
September 2014

Caffeine and adenosine A(2A) receptor inactivation decrease striatal neuropathology in a lentiviral-based model of Machado-Joseph disease.

Ann Neurol 2013 May 26;73(5):655-66. Epub 2013 Apr 26.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Objective: Machado-Joseph disease (MJD) is a neurodegenerative disorder associated with an abnormal CAG expansion, which translates into an expanded polyglutamine tract within ataxin-3. There is no therapy to prevent or modify disease progression. Because caffeine (a nonselective adenosine receptor antagonist) and selective adenosine A2A receptor (A2A R) blockade alleviate neurodegeneration in different brain diseases, namely at early stages of another polyglutamine-related disorder such as Huntington's disease, we now tested their ability to control MJD-associated neurodegeneration.

Methods: MJD was modeled by transducing the striatum of male adult C57Bl/6 mice with lentiviral vectors encoding mutant ataxin-3 in one hemisphere and wild-type ataxin-3 in the other hemisphere (as internal control). Caffeine (1g/L) was applied through the drinking water. Mice were killed at different time points (from 2 to 12 weeks) to probe for the appearance of different morphological changes using immunohistochemical analysis.

Results: Mutant ataxin-3 caused an evolving neuronal dysfunction (loss of DARPP-32 staining) leading to neurodegeneration (cresyl violet and neuronal nuclei staining) associated with increased number of mutant ataxin-3 inclusions in the basal ganglia. Notably, mutant ataxin-3 triggered early synaptotoxicity (decreased synaptophysin and microtubule-associated protein-2 staining) and reactive gliosis (glial fibrillary acidic protein and CD11b staining), which predated neuronal dysfunction and damage. Caffeine reduced the appearance of all these morphological modifications, which were also abrogated in mice with a global A2A R inactivation (knockout).

Interpretation: Our findings provide a demonstration that synaptotoxicity and gliosis are precocious events in MJD and that caffeine and A2A R inactivation decrease MJD-associated striatal pathology, which paves the way to consider A2A Rs as novel therapeutic targets to manage MJD.
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http://dx.doi.org/10.1002/ana.23866DOI Listing
May 2013

Potentiation of radiotherapy by a localized antiangiogenic gene therapy.

Radiother Oncol 2013 May 23;107(2):252-8. Epub 2013 Apr 23.

Université Catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, Brussels, Belgium.

Background And Purpose: We hypothesized that electrotransfer of a plasmid encoding an antiangiogenic factor, the recombinant disintegrin domain of ADAM-15, (pRDD) could modify the tumor microenvironment and radiosensitize tumor.

Materials And Methods: pRDD was injected in the TLT tumor or FSaII fibrosarcomas before electroporation. pO2 in tumors and oxygen consumption in vitro were measured by electronic paramagnetic resonance (EPR) oximetry. Tumor perfusion was assessed by laser doppler imaging and patent blue assay.

Results: pRDD electrotransfer caused a significant delay in TLT growth and an anti-angiogenic effect. It significantly increased tumor pO2 in TLT and FSaII for at least 4 days. pRDD electrotransfer and radiotherapy were more effective than either treatment alone. Modifications of tumor microenvironment were evaluated: tumor perfusion and interstitial fluid pressure were not modified. Oxygen consumption by the cells was decreased resulting both from a decrease in oxygen consumption rate and from a decrease in cell viability.

Conclusion: The combination of localized antiangiogenic gene therapy and radiotherapy applied in the time of maximal oxygenation could be a promising alternative for cancer treatment.
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http://dx.doi.org/10.1016/j.radonc.2013.03.018DOI Listing
May 2013

Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia.

J Neuroinflammation 2013 Jan 30;10:16. Epub 2013 Jan 30.

Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, Coimbra 3004-517, Portugal.

Background: Brain-derived neurotrophic factor (BDNF) has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs) control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation.

Methods: Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL) in the absence or in the presence of the A2AR antagonist, SCH58261 (50 nM), as well as other modulators of A2AR signaling. The BDNF cellular content and secretion were quantified by Western blotting and ELISA, A2AR density was probed by Western blotting and immunocytochemistry and cell proliferation was assessed by BrdU incorporation. Additionally, the A2AR modulation of LPS-driven cell proliferation was also tested in primary cultures of mouse microglia.

Results: LPS induced time-dependent changes of the intra- and extracellular levels of BDNF and increased microglial proliferation. The maximal LPS-induced BDNF release was time-coincident with an LPS-induced increase of the A2AR density. Notably, removing endogenous extracellular adenosine or blocking A2AR prevented the LPS-mediated increase of both BDNF secretion and proliferation, as well as exogenous BDNF-induced proliferation.

Conclusions: We conclude that A2AR activation plays a mandatory role controlling the release of BDNF from activated microglia, as well as the autocrine/paracrine proliferative role of BDNF.
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http://dx.doi.org/10.1186/1742-2094-10-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567964PMC
January 2013

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Brain 2012 Aug;135(Pt 8):2428-39

Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.
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http://dx.doi.org/10.1093/brain/aws177DOI Listing
August 2012