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Accurate assessment of post-stroke deficits is crucial in translational research. Recent advances in machine learning offer precise quantification of rodent motor behavior post-stroke, yet detecting lesion-specific upper extremity deficits remains unclear. Employing proximal middle cerebral artery occlusion (MCAO) and cortical photothrombosis (PT) in mice, we assessed post-stroke impairments via the Staircase test. Lesion locations were identified using 7 T-MRI. Machine learning was applied to reconstruct forepaw kinematic trajectories and feature analysis was achieved with MouseReach, a new data-processing toolbox. Lesion reconstructions pinpointed ischemic centers in the striatum (MCAO) and sensorimotor cortex (PT). Pellet retrieval alterations were observed, but were unrelated to overall stroke volume. Instead, forepaw slips and relative reaching success correlated with increasing cortical lesion size in both models. Striatal lesion size after MCAO was associated with prolonged reach durations that occurred with delayed symptom onset. Further analysis on the impact of selective serotonin reuptake inhibitors in the PT model revealed no clear treatment effects but replicated strong effect sizes of slips for post-stroke deficit detection. In summary, refined movement analysis unveiled specific deficits in two widely-used mouse stroke models, emphasizing the value of deep behavioral profiling in preclinical stroke research to enhance model validity for clinical translation.
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Modelos Animais de Doenças , Infarto da Artéria Cerebral Média , Acidente Vascular Cerebral , Animais , Camundongos , Masculino , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/complicações , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/complicações , Camundongos Endogâmicos C57BL , Imageamento por Ressonância Magnética/métodos , Aprendizado de Máquina , Movimento/fisiologiaRESUMO
Hemorrhagic shock (HS), a leading cause of preventable death, is characterized by severe blood loss and inadequate tissue perfusion. Reoxygenation of ischemic tissues exacerbates organ damage through ischemia-reperfusion injury. SUMOylation has been shown to protect neurons after stroke and is upregulated in response to cellular stress. However, the role of SUMOylation in organ protection after HS is unknown. This study aimed to investigate SUMOylation-mediated organ protection following HS. Male Wistar rats were subjected to HS (blood pressure of 40 ± 2 mmHg, for 90 min) followed by reperfusion. Blood, kidney, and liver samples were collected at various time points after reperfusion to assess organ damage and investigate the profile of SUMO1 and SUMO2/3 conjugation. In addition, human kidney cells (HK-2), treated with the SUMOylation inhibitor TAK-981 or overexpressing SUMO proteins, were subjected to oxygen and glucose deprivation to investigate the role of SUMOylation in hypoxia/reoxygenation injury. The animals presented progressive multiorgan dysfunction, except for the renal system, which showed improvement over time. Compared to the liver, the kidneys displayed distinct patterns in terms of oxidative stress, apoptosis activation, and tissue damage. The global level of SUMO2/3 in renal tissue was also distinct, suggesting a differential role. Pharmacological inhibition of SUMOylation reduced cell viability after hypoxia-reoxygenation damage, while overexpression of SUMO1 or SUMO2 protected the cells. These findings suggest that SUMOylation might play a critical role in cellular protection during ischemia-reperfusion injury in the kidneys, a role not observed in the liver. This difference potentially explains the renal resilience observed in HS animals when compared to other systems.
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Ratos Wistar , Choque Hemorrágico , Sumoilação , Animais , Masculino , Choque Hemorrágico/metabolismo , Sumoilação/efeitos dos fármacos , Sumoilação/fisiologia , Ratos , Humanos , Rim/metabolismo , Rim/patologia , Rim/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Linhagem CelularRESUMO
BACKGROUND: Prediction of poststroke outcome using the degree of subacute deficit or magnetic resonance imaging is well studied in humans. While mice are the most commonly used animals in preclinical stroke research, systematic analysis of outcome predictors is lacking. METHODS: We intended to incorporate heterogeneity into our retrospective study to broaden the applicability of our findings and prediction tools. We therefore analyzed the effect of 30, 45, and 60 minutes of arterial occlusion on the variance of stroke volumes. Next, we built a heterogeneous cohort of 215 mice using data from 15 studies that included 45 minutes of middle cerebral artery occlusion and various genotypes. Motor function was measured using a modified protocol for the staircase test of skilled reaching. Phases of subacute and residual deficit were defined. Magnetic resonance images of stroke lesions were coregistered on the Allen Mouse Brain Atlas to characterize stroke topology. Different random forest prediction models that either used motor-functional deficit or imaging parameters were generated for the subacute and residual deficits. RESULTS: Variance of stroke volumes was increased by 45 minutes of arterial occlusion compared with 60 minutes. The inclusion of various genotypes enhanced heterogeneity further. We detected both a subacute and residual motor-functional deficit after stroke in mice and different recovery trajectories could be observed. In mice with small cortical lesions, lesion volume was the best predictor of the subacute deficit. The residual deficit could be predicted most accurately by the degree of the subacute deficit. When using imaging parameters for the prediction of the residual deficit, including information about the lesion topology increased prediction accuracy. A subset of anatomic regions within the ischemic lesion had particular impact on the prediction of long-term outcomes. Prediction accuracy depended on the degree of functional impairment. CONCLUSIONS: For the first time, we developed and validated a robust tool for the prediction of functional outcomes after experimental stroke in mice using a large and genetically heterogeneous cohort. These results are discussed in light of study design and imaging limitations. In the future, using outcome prediction can improve the design of preclinical studies and guide intervention decisions.
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The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.
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Magnetic resonance imaging (MRI) is widely used for ischemic stroke lesion detection in mice. A challenge is that lesion segmentation often relies on manual tracing by trained experts, which is labor-intensive, time-consuming, and prone to inter- and intra-rater variability. Here, we present a fully automated ischemic stroke lesion segmentation method for mouse T2-weighted MRI data. As an end-to-end deep learning approach, the automated lesion segmentation requires very little preprocessing and works directly on the raw MRI scans. We randomly split a large dataset of 382 MRI scans into a subset (n = 293) to train the automated lesion segmentation and a subset (n = 89) to evaluate its performance. We compared Dice coefficients and accuracy of lesion volume against manual segmentation, as well as its performance on an independent dataset from an open repository with different imaging characteristics. The automated lesion segmentation produced segmentation masks with a smooth, compact, and realistic appearance that are in high agreement with manual segmentation. We report dice scores higher than the agreement between two human raters reported in previous studies, highlighting the ability to remove individual human bias and standardize the process across research studies and centers.
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Aprendizado Profundo , AVC Isquêmico , Trabalho de Parto , Acidente Vascular Cerebral , Humanos , Gravidez , Feminino , Animais , Camundongos , Acidente Vascular Cerebral/diagnóstico por imagem , Imageamento por Ressonância MagnéticaRESUMO
Paracrine cerebral Interleukin 6 (Il6) is relevant for stroke recovery, but systemic Il6 elevation may worsen outcome. Hence, paracrine Il6 response modulation within the neurovascular unit has emerged as an attractive therapeutic approach. Lithium modulates Il6 responses and improves stroke outcome. However, lithium may cause serious adverse effects. Here, we report that Zincfinger protein 580 (Zfp580) mediates the effects of lithium on Il6 signaling. In contrast to lithium, Zfp580 inactivation had no neurotoxic effects, and Zfp580 knock out mice showed no phenotypic changes in cognitive and motor function behavioral tests. We discovered that lithium and hypoxia disinhibited Il6 via Zfp580 suppression and post-translational modification by small ubiquitin-like modifier (SUMO). After transient middle cerebral artery occlusion, loss of Zfp580 reduced paracrine Il6 and increased Il6 trans-signaling. Aside from modulating Il6 signaling, Zfp580 loss improved endothelial resilience to ischemia, was highly neuroprotective resulting in smaller infarcts and enhanced use-dependent neuroplasticity, all of which led to improved functional outcome. In conclusion, inactivation of Zfp580 exerts positive effects on multiple key mechanisms without exhibiting relevant adverse side effects, making it potentially a more specific and effective treatment target for stroke recovery than lithium. To fully assess its potential, Zfp580 inhibitors must be developed.
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Isquemia Encefálica , Acidente Vascular Cerebral , Camundongos , Animais , Interleucina-6 , Lítio , Fatores de Transcrição/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Transdução de SinaisRESUMO
BACKGROUND: DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumours. In fact, DNA methylation profiling of human brain tumours already profoundly impacts clinical neuro-oncology. However, current implementation using hybridisation microarrays is time consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification. Here, we demonstrate that this approach allows us to discriminate a wide spectrum of primary brain tumours. RESULTS: Using public reference data of 82 distinct tumour entities, we performed nanopore genome sequencing on 382 tissue samples covering 46 brain tumour (sub)types. Using bootstrap sampling in a cohort of 55 cases, we found that a minimum set of 1000 random CpG features is sufficient for high-confidence classification by ad hoc random forests. We implemented score recalibration as a confidence measure for interpretation in a clinical context and empirically determined a platform-specific threshold in a randomly sampled discovery cohort (N = 185). Applying this cut-off to an independent validation series (n = 184) yielded 148 classifiable cases (sensitivity 80.4%) and demonstrated 100% specificity. Cross-lab validation demonstrated robustness with concordant results across four laboratories in 10/11 (90.9%) cases. In a prospective benchmarking (N = 15), the median time to results was 21.1 h. CONCLUSIONS: In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumours. Platform-specific confidence scores facilitate clinical implementation for which prospective evaluation is warranted and ongoing.
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Neoplasias Encefálicas , Sequenciamento por Nanoporos , Humanos , Metilação de DNA , Neoplasias Encefálicas/patologia , GenomaRESUMO
Clonal hematopoiesis (CH) is common among older people and is associated with an increased risk of atherosclerosis, inflammation, and shorter overall survival. Age and inflammation are major risk factors for ischemic stroke, yet the association of CH with risk of secondary vascular events and death is unknown. We investigated CH in peripheral blood DNA from 581 patients with first-ever ischemic stroke from the Prospective Cohort With Incident Stroke-Berlin study using error-corrected targeted sequencing. The primary composite end point (CEP) consisted of recurrent stroke, myocardial infarction, and all-cause mortality. A total of 348 somatic mutations with a variant allele frequency ≥1% were identified in 236 of 581 patients (41%). CH was associated with large-artery atherosclerosis stroke (P = .01) and white matter lesion (P < .001). CH-positive patients showed increased levels of proinflammatory cytokines, such as interleukin-6 (IL-6), interferon gamma, high-sensitivity C-reactive protein, and vascular cell adhesion molecule 1. CH-positive patients had a higher risk for the primary CEP (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.04-2.31; P = .03), which was more pronounced in patients with larger clones. CH clone size remained an independent risk factor (HR, 1.30; 95% CI, 1.04-1.62; P = .022) in multivariable Cox regression. Although our data show that, in particular, larger and TET2- or PPM1D-mutated clones are associated with increased risk of recurrent vascular events and death, this risk is partially mitigated by a common germline variant of the IL-6 receptor (IL-6R p.D358A). The CH mutation profile is accompanied by a proinflammatory profile, opening new avenues for preventive precision medicine approaches to resolve the self-perpetuating cycle of inflammation and clonal expansion.
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Aterosclerose , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Idoso , Hematopoiese Clonal/genética , Estudos Prospectivos , Hematopoese/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/complicações , Inflamação/genética , Inflamação/complicações , Aterosclerose/complicações , MutaçãoRESUMO
A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.
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Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace-solid phase microextraction-gas chromatography-mass spectrometry-olfactometry (HS-SPME-GC-MS-O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò-Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time. KEY POINTS: ⢠Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes. ⢠Odors of several Z-unsaturated fatty aldehydes are described for the first time. ⢠Potential for industrial production of aldehyde-based odorants from natural sources.
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Dioxigenases , Odorantes , Aldeídos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Odorantes/análiseRESUMO
SUMOylation is described as a posttranslational protein modification (PTM) that is involved in the pathophysiological processes underlying several conditions related to ischemia- and reperfusion-induced damage. Increasing evidence suggests that, under low oxygen levels, SUMOylation might be part of an endogenous mechanism, which is triggered by injury to protect cells within the central nervous system. However, the role of ischemia-induced SUMOylation in the periphery is still unclear. This article summarizes the results of recent studies regarding SUMOylation profiles in several diseases characterized by impaired blood flow to the cardiorenal, gastrointestinal, and respiratory systems. Our review shows that although ischemic injury per se does not always increase SUMOylation levels, as seen in strokes, it seems that in most cases the positive modulation of protein SUMOylation after peripheral ischemia might be a protective mechanism. This complex relationship warrants further investigation, as the role of SUMOylation during hypoxic conditions differs from organ to organ and is still not fully elucidated.
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Processamento de Proteína Pós-Traducional , Sumoilação , PerfusãoRESUMO
Insulin-like growth factor 1 (Igf1) and insulin-like growth factor binding protein 3 (Igfbp3) are endocrine and paracrine factors that influence stroke occurrence, severity, and recovery. Low levels of endocrine Igf1 and Igfbp3 were associated with larger infarct volumes and unfavorable outcomes. Paracrine Igf1 is brain cytoprotective and improves functional recovery after stroke. In this study, we evaluated the effects of zinc finger protein 580 (Zfp580) on endocrine and paracrine Igf1 and Igfbp3 after stroke. Zfp580 suppressed the expression of Igf1 and Igfbp3 in cerebral microvascular endothelial cells (bEnd.3) as determined by real-time RT-PCR. Zfp580 was suppressed by combined oxygen and glucose deprivation (OGD) and mediated the effect of OGD on Igf1 and Igfbp3. In vivo, we evaluated paracrine regulation by real-time RT-PCR of brain lysates and endocrine regulation by ELISA of blood samples. Genomic ablation of Zfp580 did not alter basal paracrine or endocrine Igf1 and Igfbp3 levels. After transient middle cerebral artery occlusion (MCAo), Zfp580 was globally elevated in the brain for up to 3 days. Paracrine Igf1 and Igfbp3 were selectively induced in the ischemic hemisphere from day 2 to day 3 or day 1 to day 7, respectively. In Zfp580 knockout mice, the paracrine regulations of Igf1 and Igfbp3 were attenuated while endocrine Igf1 and the molar Igf1/Igfbp3 ratio were increased. In conclusion, Zfp580 differentially controls paracrine and endocrine Igf1 and Igfbp3 after stroke. Inhibition of Zfp580 might be a new treatment target leading to increased activity of Igf1 to improve stroke outcome.
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Ischemic diseases are the leading cause of death and disability worldwide. The main compensatory mechanism by which our body responds to reduced or blocked blood flow caused by ischemia is mediated by collateral vessels. Collaterals are present in many healthy tissues (including brain and heart) and serve as natural bypass vessels, by bridging adjacent arterial trees. This review focuses on: the definition and significance of pial collateral vessels, the described mechanism of pial collateral formation, an overview of molecular players and pathways involved in pial collateral biology and emerging approaches to prevent or mitigate risk factor-associated loss of pial collaterals. Despite their high clinical relevance and recent scientific efforts toward understanding collaterals, much of the fundamental biology of collaterals remains obscure.
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Gait impairments in Parkinson's disease remain a scientific and therapeutic challenge. The advent of new deep brain stimulation (DBS) devices capable of recording brain activity from chronically implanted electrodes has fostered new studies of gait in freely moving patients. The hope is to identify gait-related neural biomarkers and improve therapy using closed-loop DBS. In this context, animal models offer a wealth of opportunities to investigate gait network impairments at multiple biological scales and address unresolved questions from clinical research. Yet, the contribution of rodent models to the development of future neuromodulation therapies will rely on translational validity. In this review, we summarize the most effective strategies to model parkinsonian gait in rodents. We discuss how clinical observations have inspired targeted brain lesions in animal models, and whether resulting motor deficits and network oscillations match recent findings in humans. We conclude that future research should incorporate behavioral tests with increased cognitive demands to potentially uncover episodic gait impairments in rodents. Additionally, we expect that basic research will benefit from the implementation of evolving signal processing strategies from clinical research. This coevolution of translational research may contribute to the future optimization of gait therapy in Parkinson's disease.
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Estimulação Encefálica Profunda , Transtornos Neurológicos da Marcha , Doença de Parkinson , Animais , Estimulação Encefálica Profunda/métodos , Marcha/fisiologia , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/terapia , Humanos , Doença de Parkinson/complicações , Doença de Parkinson/patologia , Doença de Parkinson/terapia , RoedoresRESUMO
BACKGROUND: Connectome analysis of neuroimaging data is a rapidly expanding field that offers the potential to diagnose, characterize, and predict neurological disease. Animal models provide insight into biological mechanisms that underpin disease, but connectivity approaches are currently lagging in the rodent. METHODS: We present a pipeline adapted for structural and functional connectivity analysis of the mouse brain, and we tested it in a mouse model of vascular dementia. RESULTS: We observed lacunar infarctions, microbleeds, and progressive white matter change across 6 months. For the first time, we report that default mode network activity is disrupted in the mouse model. We also identified specific functional circuitry that was vulnerable to vascular stress, including perturbations in a sensorimotor, visual resting state network that were accompanied by deficits in visual and spatial memory tasks. CONCLUSIONS: These findings advance our understanding of the mouse connectome and provide insight into how it can be altered by vascular insufficiency.
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Conectoma , Demência Vascular , Animais , Encéfalo/diagnóstico por imagem , Conectoma/métodos , Demência Vascular/diagnóstico por imagem , Modelos Animais de Doenças , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Rede NervosaRESUMO
Deep brain stimulation (DBS) is the preferred treatment for therapy-resistant movement disorders such as dystonia and Parkinson's disease (PD), mostly in advanced disease stages. Although DBS is already in clinical use for ~30 years and has improved patients' quality of life dramatically, there is still limited understanding of the underlying mechanisms of action. Rodent models of PD and dystonia are essential tools to elucidate the mode of action of DBS on behavioral and multiscale neurobiological levels. Advances have been made in identifying DBS effects on the central motor network, neuroprotection and neuroinflammation in DBS studies of PD rodent models. The phenotypic dtsz mutant hamster and the transgenic DYT-TOR1A (ΔETorA) rat proved as valuable models of dystonia for preclinical DBS research. In addition, continuous refinements of rodent DBS technologies are ongoing and have contributed to improvement of experimental quality. We here review the currently existing literature on experimental DBS in PD and dystonia models regarding the choice of models, experimental design, neurobiological readouts, as well as methodological implications. Moreover, we provide an overview of the technical stage of existing DBS devices for use in rodent studies.
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Estimulação Encefálica Profunda/métodos , Modelos Animais de Doenças , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/terapia , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cricetinae , Humanos , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/patologia , Ratos , RoedoresRESUMO
Tumor-endothelial cell interactions represent an essential mechanism in spinal metastasis. Ephrin-B2-EphB4 communication induces tumor cell repulsion from the endothelium in metastatic melanoma, reducing spinal bone metastasis formation. To shed further light on the Ephrin-B2-EphB4 signaling mechanism, we researched the effects of pharmacological EphB4 receptor stimulation and inhibition in a ligand-dependent/independent context. We chose a preventative and a post-diagnostic therapeutic window. EphB4 stimulation during tumor cell seeding led to an increase in spinal metastatic loci and number of disseminated melanoma cells, as well as earlier locomotion deficits in the presence of endothelial Ephrin-B2. In the absence of endothelial Ephrin-B2, reduction of metastatic loci with a later manifestation of locomotion deficits occurred. Thus, EphB4 receptor stimulation affects metastatic dissemination depending on the presence/absence of endothelial Ephrin-B2. After the manifestation of solid metastasis, EphB4 kinase inhibition resulted in significantly earlier manifestation of locomotion deficits in the presence of the ligand. No post-diagnostic treatment effect was found in the absence of endothelial Ephrin-B2. For solid metastasis treatment, EphB4 kinase inhibition induced prometastatic effects in the presence of endothelial Ephrin-B2. In the absence of endothelial Ephrin-B2, both therapies showed no effect on the growth of solid metastasis.
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Efrina-B2/metabolismo , Melanoma Experimental/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor EphB4/metabolismo , Transdução de Sinais , Neoplasias da Coluna Vertebral/metabolismo , Animais , Linhagem Celular Tumoral , Efrina-B2/genética , Ligantes , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Receptor EphB4/genética , Neoplasias da Coluna Vertebral/tratamento farmacológico , Neoplasias da Coluna Vertebral/genética , Neoplasias da Coluna Vertebral/secundárioRESUMO
Serotonin (5-HT) is one of the major neuromodulators present in the mammalian brain and has been shown to play a role in multiple physiological processes. The mechanisms by which 5-HT modulates cortical network activity, however, are not yet fully understood. We investigated the effects of 5-HT on slow oscillations (SOs), a synchronized cortical network activity universally present across species. SOs are observed during anesthesia and are considered to be the default cortical activity pattern. We discovered that (±)3,4-methylenedioxymethamphetamine (MDMA) and fenfluramine, two potent 5-HT releasers, inhibit SOs within the entorhinal cortex (EC) in anesthetized mice. Combining opto- and pharmacogenetic manipulations with in vitro electrophysiological recordings, we uncovered that somatostatin-expressing (Sst) interneurons activated by the 5-HT2A receptor (5-HT2AR) play an important role in the suppression of SOs. Since 5-HT2AR signaling is involved in the etiology of different psychiatric disorders and mediates the psychological effects of many psychoactive serotonergic drugs, we propose that the newly discovered link between Sst interneurons and 5-HT will contribute to our understanding of these complex topics.
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Córtex Entorrinal/fisiologia , Interneurônios/fisiologia , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/metabolismo , Animais , CamundongosRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by a late clinical onset of psychiatric, cognitive, and motor symptoms. Transcriptional dysregulation is an early and central disease mechanism which is accompanied by epigenetic alterations in HD. Previous studies demonstrated that targeting transcriptional changes by inhibition of histone deacetylases (HDACs), especially the class I HDACs, provides therapeutic effects. Yet, their exact mechanisms of action and the features of HD pathology, on which these inhibitors act remain to be elucidated. Here, using transcriptional profiling, we found that selective inhibition of HDAC1 and HDAC3 by RGFP109 alleviated transcriptional dysregulation of a number of genes, including the transcription factor genes Neurod2 and Nr4a2, and gene sets and programs, especially those that are associated to insulin-like growth factor pathway, in the striatum of R6/1 mice. RGFP109 treatment led to a modest improvement of the motor skill learning and coordination deficit on the RotaRod test, while it did not alter the locomotor and anxiety-like phenotypes in R6/1 animals. We also found, by volumetric MRI, a widespread brain atrophy in the R6/1 mice at the symptomatic disease stage, on which RGFP109 showed no significant effects. Collectively, our combined work suggests that specific HDAC1 and HDAC3 inhibition may offer benefits for alleviating the motor phenotypic deficits and transcriptional dysregulation in HD.
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Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. However, despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have not been fully uncovered. This limits the options for pharmacological treatment of glutamate-related motor disorders, including Huntington's disease (HD). We therefore investigated the possible binding partners of transgenic EAAT2 and their alterations under the influence of mutant huntingtin (mHTT). Mass spectrometry analysis after pull-down of striatal YFP-EAAT2 from wild-type (WT) mice and heterozygote (HET) Q175 mHTT-knock-in mice identified a total of 148 significant (FDR < 0.05) binders to full-length EAAT2. Of them 58 proteins exhibited mHTT-related differences. Most important, in 26 of the 58 mHTT-sensitive cases, protein abundance changed back toward WT levels when the mice expressed a C-terminal-truncated instead of full-length variant of EAAT2. These findings motivated new attempts to clarify the role of astrocytic EAAT2 regulation in cortico-basal movement control. Striatal astrocytes of Q175 HET mice were targeted by a PHP.B vector encoding EAAT2 with different degree of C-terminal modification, i.e., EAAT2-S506X (truncation at S506), EAAT2-4KR (4 lysine to arginine substitutions) or EAAT2 (full-length). The results were compared to HET and WT injected with a tag-only vector (CTRL). It was found that the presence of a C-terminal-modified EAAT2 transgene (i) increased the level of native EAAT2 protein in striatal lysates and perisynaptic astrocyte processes, (ii) enhanced the glutamate uptake of transduced astrocytes, (iii) stimulated glutamate clearance at individual corticostriatal synapses, (iv) increased the glutamate uptake of striatal astrocytes and (iv) alleviated the mHTT-related hypokinesia (open field indicators of movement initiation). In contrast, over-expression of full-length EAAT2 neither facilitated glutamate uptake nor locomotion. Together, our results support the new hypothesis that preventing abnormal protein-protein interactions at the C-terminal of EAAT2 could eliminate the mHTT-related deficits in corticostriatal synaptic glutamate clearance and movement initiation.