RESUMO
Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and CSF, which is mainly secreted by liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS. In TTR deficient mice, we observed that anti-TTR Nanobody was successfully distributed throughout all brain areas, and also reaching the spinal cord. In wild-type mice, a similar distribution pattern was observed. However, in areas known to be rich in TTR, reduced levels of Nanobody were found, suggesting potential target-mediated effects. Indeed, in wild-type mice, the anti-TTR Nanobody was specifically internalized in a receptor-mediated process, by neuronal-like cells, which were identified as motor neurons. Whereas in KO TTR mice Nanobody was internalized by all cells, for late lysosomal degradation. Moreover, we demonstrate that in vivo motor neurons also actively synthesize TTR. Finally, in vitro cultured primary motor neurons were also found to synthesize and secrete TTR into culture media. Thus, through a novel intranasal CNS distribution study with an anti-TTR Nanobody, we disclose a new cell type capable of synthesizing TTR, which might be important for the understanding of the physiological role of TTR, as well as in pathological conditions where TTR levels are altered in CSF, such as amyotrophic lateral sclerosis.
Assuntos
Encéfalo/metabolismo , Neurônios Motores/metabolismo , Pré-Albumina/metabolismo , Medula Espinal/metabolismo , Administração Intranasal , Animais , Camundongos , Camundongos Knockout , Anticorpos de Domínio Único/administração & dosagemRESUMO
Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal survival through activation of TrkB receptors. The trkB gene encodes a full-length receptor tyrosine kinase (TrkB.FL) and its truncated (T1/T2) isoforms. We investigated the changes in TrkB protein levels and signaling activity under excitotoxic conditions, which are characteristic of brain ischemia, traumatic brain injury, and neurodegenerative disorders. Excitotoxic stimulation of cultured rat hippocampal or striatal neurons downregulated TrkB.FL and upregulated a truncated form of the receptor (TrkB.T). Downregulation of TrkB.FL was mediated by calpains, whereas the increase in TrkB.T protein levels required transcription and translation activities. Downregulation of TrkB.FL receptors in hippocampal neurons correlated with a decrease in BDNF-induced activation of the Ras/ERK and PLCγ pathways. However, calpain inhibition, which prevents TrkB.FL degradation, did not preclude the decrease in signaling activity of these receptors. On the other hand, incubation with anisomycin, to prevent the upregulation of TrkB.T, protected to a large extent the TrkB.FL signaling activity, suggesting that truncated receptors may act as dominant-negatives. The upregulation of TrkB.T under excitotoxic conditions was correlated with an increase in BDNF-induced inhibition of RhoA, a mediator of excitotoxic neuronal death. BDNF fully protected hippocampal neurons transduced with TrkB.T when present during excitotoxic stimulation with glutamate, in contrast with the partial protection observed in cells overexpressing TrkB.FL or expressing GFP. These results indicate that BDNF protects hippocampal neurons by two distinct mechanisms: through the neurotrophic effects of TrkB.FL receptors and by activation of TrkB.T receptors coupled to inhibition of the excitotoxic signaling.
Assuntos
Morte Celular/efeitos dos fármacos , Corpo Estriado/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Caínico/toxicidade , Receptor trkB/metabolismo , Animais , Anisomicina/farmacologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Calpaína/antagonistas & inibidores , Calpaína/fisiologia , Morte Celular/genética , Corpo Estriado/efeitos dos fármacos , Embrião de Mamíferos , Ácido Glutâmico/toxicidade , Hipocampo/efeitos dos fármacos , Isoenzimas/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Cultura Primária de Células , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.
Assuntos
Neurotoxinas/farmacologia , Sinapses/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Animais , Western Blotting , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Calpaína/metabolismo , DNA/genética , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/patologia , Ácido Caínico/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , Monoéster Fosfórico Hidrolases/metabolismo , Plasmídeos/genética , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Sinapses/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Transfecção , Ácido gama-Aminobutírico/fisiologiaRESUMO
Ischemic stroke is a leading cause of death worldwide, mainly in western countries. So far, approved therapies rely on reperfusion of the affected brain area, by intravenous thrombolysis or mechanical thrombectomy. The last approach constitutes a breakthrough in the field, by extending the therapeutic window to 16-24 h after stroke onset and reducing stroke mortality. The combination of pharmacological brain-protective strategies with reperfusion is the future of stroke therapy, aiming to reduce brain cell death and decrease patients' disabilities. Recently, a brain-protective drug-nerinetide-reduced brain infarct and stroke mortality, and improved patients' functional outcomes in clinical trials. The success of new therapies relies on bringing preclinical studies and clinical practice close together, by including a functional outcome assessment similar to clinical reality. In this review, we focused on recent upgrades of in vitro and in vivo stroke models for more accurate and effective evaluation of therapeutic strategies: from spheroids to organoids, in vitro models that include all brain cell types and allow high throughput drug screening, to advancements in in vivo preclinical mouse stroke models to mimic the clinical reality in surgical procedures, postsurgical care, and functional assessment.
RESUMO
Acute ischemic stroke (AIS) remains a leading cause of mortality, despite significant advances in therapy (endovascular thrombectomy). Failure in developing novel effective therapies is associated with unsuccessful translation from preclinical studies to clinical practice, associated to inconsistent and highly variable infarct areas and lack of relevant post-stroke functional evaluation in preclinical research. To outreach these limitations, we optimized the intraluminal transient middle cerebral occlusion, a widely used mouse stroke model, in two key parameters, selection of appropriate occlusion filaments and time of occlusion, which show a significant variation in the literature. We demonstrate that commercially available filaments with short coating length (1-2 mm), together with 45-min occlusion, results in a consistent affected brain region, similar to what is observed in most patients with AIS. Importantly, a dedicated post-stroke care protocol, based on clinical practice applied to patients who had stroke, resulted in lower mortality and improved mice welfare. Finally, a battery of tests covering relevant fine motor skills, sensory functions, and learning/memory behaviors revealed a significant effect of tMCAO brain infarction, which is parallel to patient symptomatology as measured by relevant clinical scales (NIH Stroke Scale, NIHSS and modified Rankin Scale, mRS). Thus, in order to enhance translation to clinical practice, future preclinical stroke research must consider the methodology described in this study, which includes improved reproducible surgical procedure, postoperative care, and the battery of functional tests. This will be a major step s closing the gap from bench to bedside, rendering the development of novel effective therapeutic approaches.
RESUMO
Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.
Assuntos
Iodo , Acidente Vascular Cerebral , Animais , Camundongos , Tetróxido de Ósmio , Microtomografia por Raio-X , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/patologia , Modelos Animais de DoençasRESUMO
Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels.
Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Neurônios/efeitos dos fármacos , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Calpaína/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Embrião de Mamíferos , Glucose/deficiência , Hipocampo/citologia , Hipóxia/patologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Ratos , Ratos Wistar , Vesículas Sinápticas/efeitos dos fármacos , Transfecção , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
Donnai-Barrow syndrome, a genetic disorder associated to LRP2 (low-density lipoprotein receptor 2/megalin) mutations, is characterized by unexplained neurological symptoms and intellectual deficits. Megalin is a multifunctional endocytic clearance cell-surface receptor, mostly described in epithelial cells. This receptor is also expressed in the CNS, mainly in neurons, being involved in neurite outgrowth and neuroprotective mechanisms. Yet, the mechanisms involved in the regulation of megalin in the CNS are poorly understood. Using transthyretin knockout mice, a megalin ligand, we found that transthyretin positively regulates neuronal megalin levels in different CNS areas, particularly in the hippocampus. Transthyretin is even able to rescue megalin downregulation in transthyretin knockout hippocampal neuronal cultures, in a positive feedback mechanism via megalin. Importantly, transthyretin activates a regulated intracellular proteolysis mechanism of neuronal megalin, producing an intracellular domain, which is translocated to the nucleus, unveiling megalin C-terminal as a potential transcription factor, able to regulate gene expression. We unveil that neuronal megalin reduction affects physiological neuronal activity, leading to decreased neurite number, length and branching, and increasing neuronal susceptibility to a toxic insult. Finally, we unravel a new unexpected role of megalin in synaptic plasticity, by promoting the formation and maturation of dendritic spines, and contributing for the establishment of active synapses, both in in vitro and in vivo hippocampal neurons. Moreover, these structural and synaptic roles of megalin impact on learning and memory mechanisms, since megalin heterozygous mice show hippocampal-related memory and learning deficits in several behaviour tests. Altogether, we unveil a complete novel role of megalin in the physiological neuronal activity, mainly in synaptic plasticity with impact in learning and memory. Importantly, we contribute to disclose the molecular mechanisms underlying the cognitive and intellectual disabilities related to megalin gene pathologies.
RESUMO
BDNF plays a key role in neuronal development, in short- and long-term changes in synaptic activity, and in neuronal survival. These effects are mediated, to a great extent, by changes in protein synthesis. We conducted a gel-based proteome profiling of the long-term (12 h) effects of BDNF in cultured hippocampal neurons. BDNF changed the abundance of proteins involved in (i) Nucleobase, nucleoside, nucleotide and nucleic acid metabolism, (ii) protein metabolism, (iii) carbohydrate metabolism, (iv) regulators of apoptosis, and (v) regulators of cell proliferation. A large majority of the identified proteins involved in translation activity were upregulated, but not all changes in the protein content were correlated with alterations in the corresponding mRNA. The upregulation of Seryl-aminoacyl-tRNA-synthetase and Eef2 was sensitive to the mTOR inhibitor rapamycin, as determined by Western blot. Since the mRNAs for proteins involved in translation represent a large fraction of the diversity of dendritic mRNAs, we investigated the effect of BDNF on the distribution of the transcripts in the soma versus neurite compartments. The increase in mRNA for proteins of the translation machinery in the soma was differentially coupled with the upregulation of neurite transcripts. BDNF also downregulated specific mRNAs in neurite compartments suggesting that the neurotrophin may act by regulating mRNA stability and thereby affecting the dendritic protein content.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dendritos/metabolismo , Hipocampo/metabolismo , Proteoma/metabolismo , RNA Mensageiro/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Marcação por Isótopo , Modelos Biológicos , Fatores de Alongamento de Peptídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Biossíntese de Proteínas , Ratos , Serina-Treonina Quinases TORRESUMO
Transthyretin (TTR) has intrinsic neurotrophic physiological activities independent from its thyroxine ligands, which involve activation of signaling pathways through interaction with megalin. Still, the megalin binding motif on TTR is unknown. Nanobodies (Nb) have the ability to bind "hard to reach" epitopes being useful tools for protein/structure function. In this work, we characterize two anti-TTR Nanobodies, with similar mouse TTR binding affinities, although only one is able to block its neuritogenic activity (169F7_Nb). Through epitope mapping, we identified amino acids 14-18, at the entrance of the TTR central channel, to be important for interaction with megalin, and a stable TTR K15N mutant in that region was constructed. The TTR K15N mutant lacks neuritogenic activity, indicating that K15 is critical for TTR neuritogenic activity. Thus, we identify the putative binding site for megalin and describe two Nanobodies that will allow research and clarification of TTR physiological properties, regarding its neurotrophic effects.
Assuntos
Sítios de Ligação/efeitos dos fármacos , Epitopos/efeitos dos fármacos , Pré-Albumina/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Humanos , Ligantes , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/efeitos dos fármacos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacosRESUMO
Glial cells and neurons are in constant reciprocal signalling both under physiological and neuropathological conditions. Microglial activation is often associated with neuronal death during inflammation of the CNS, although microglial cells are also known to exert a neuroprotective role. In this work, we investigated the interplay between cerebellar granule neurons (CGN) and microglia in the perspective of CGN survival to an excitotoxic stimulus, quinolinic acid (QA), a catabolite of the tryptophan degradation pathway. We observed that CGN succumb to QA challenge via extracellular signal regulated kinase 1 and 2 (ERK) activation. Our data with transgenic mice expressing the natural inhibitor of calpains, calpastatin, indicate that together with cathepsins they mediate QA-induced toxicity acting downstream of the mitogen-activated protein kinase kinase-ERK pathway. Microglial cells are not only resistant to QA but can rescue neurons from QA-mediated toxicity when they are mixed in culture with neurons or by using mixed culture-conditioned medium (MCCM). This effect is mediated via fibroblast growth factor-2 (FGF-2) present in MCCM. FGF-2 is transcriptionally up-regulated in neurons and secreted in the MCCM as a result of neuron-microglia crosstalk. The neuroprotection is associated with the retention of cathepsins in the lysosomes and with transactivation of inducible heat-shock protein 70 downstream of FGF-2. Furthermore, FGF-2 upon release by neurons activates c-jun N-terminal kinase 1 and 2 pathway which also contributes to neuronal survival. We suggest that FGF-2 plays a pivotal role in neuroprotection against QA as an outcome of neuron-microglia interaction.
Assuntos
Citoproteção/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Microglia/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Animais , Encefalopatias/metabolismo , Encefalopatias/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Catepsinas/metabolismo , Comunicação Celular/fisiologia , Morte Celular/fisiologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Técnicas de Cultura de Órgãos , Ácido Quinolínico/metabolismo , Ácido Quinolínico/toxicidade , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system, and changes in GABAergic neurotransmission modulate the activity of neuronal networks. Gephyrin is a scaffold protein responsible for the traffic and synaptic anchoring of GABAA receptors (GABAAR); therefore, changes in gephyrin expression and oligomerization may affect the activity of GABAergic synapses. In this work, we investigated the changes in gephyrin protein levels during brain ischemia and in excitotoxic conditions, which may affect synaptic clustering of GABAAR. We found that gephyrin is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, as well as after intrahippocampal injection of kainate, giving rise to a stable cleavage product. Gephyrin cleavage was also observed in cultured hippocampal neurons subjected to transient oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, and after transient middle cerebral artery occlusion (MCAO) in mice, a model of focal brain ischemia. Furthermore, a truncated form of gephyrin decreased the synaptic clustering of the protein, reduced the synaptic pool of GABAAR containing γ2 subunits and upregulated OGD-induced cell death in hippocampal cultures. Our results show that excitotoxicity and brain ischemia downregulate full-length gephyrin with a concomitant generation of truncated products, which affect synaptic clustering of GABAAR and cell death.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Receptores de GABA-A/metabolismo , Animais , Calpaína/metabolismo , Morte Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Glucose/deficiência , Ácido Glutâmico/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Neurotoxinas/toxicidade , Oxigênio , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Transthyretin (TTR) is the carrier protein of thyroxine (T4) and binds to retinol-binding protein (RBP)-retinol complex. It is mainly synthesized by both liver and choroid plexuses of the brain. Besides these properties, it has a neuroprotective role in several contexts such as Alzheimer's disease (AD) and cerebral ischemia. Activation of insulin-like growth factor receptor I (IGF-IR) pathways and increased levels of TTR are associated with absence of neurodegeneration in an AD mouse model. In the present study, we verified that young/adult TTR null mice had decreased levels of IGF-IR in the hippocampus, but not in choroid plexus when compared with wild-type age-matched controls. Moreover, we could also demonstrate that conditional silencing of peripheral TTR did not have any influence in hippocampal IGF-IR levels, indicating that TTR effect on IGF-IR levels is due to TTR mainly synthesized in the choroid plexus. In vitro cellular studies, using NIH3T3 cell line and primary cultured hippocampal neurons, we showed that TTR upregulates IGF-IR at the transcription and translation levels and that is dependent on receptor internalization. Using a GFP-IGF-IR fusion protein, we also found that TTR triggers IGF-IR nuclear translocation in cultured neurons. We could also see an enrichment of IGF-IR in the nuclear fraction, after TTR stimulation in NIH3T3 cells, indicating that IGF-IR regulation, triggered by TTR is induced by nuclear translocation. In summary, the results provide evidence of a new role of TTR as a transcription inducer of IGF-IR in central nervous system (CNS), unveiling a new role in neuroprotection.