RESUMEN

5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used by neurosurgeons to identify the tumor cells of high-grade gliomas during operation. However, the issue of whether 5-ALA-induced PpIX fluorescence consistently stains all the tumor cells is still debated. Here, we assessed the cytoplasmatic signal of 5-ALA by fluorescence microscopy in a series of human gliomas. As tumor markers, we used antibodies against collapsin response-mediated protein 5 (CRMP5), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and anti-isocitrate dehydrogenase 1 (IDH1). In grade III-IV gliomas, the signal induced by 5-ALA was detected in 32.7-75.5 percent of CRMP5-expressing tumor cells. In low-grade gliomas (WHO grade II), the CRMP5-expressing tumor cells did not fluoresce following 5-ALA. Immunofluorescence with antibodies that stain various components of the blood-brain barrier (BBB) suggested that 5-ALA does not cross the un-breached BBB, in spite of its small dimension. To conclude, 5-ALA-induced PpIX fluorescence has an established role in high-grade glioma surgery, but it has limited usefulness in surgery for low-grade glioma, especially when the BBB is preserved.

RESUMEN

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-α) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation.

Asunto(s)

Neoplasias Encefálicas/patología , Diferenciación Celular/fisiología , Glioma/patología , Recurrencia Local de Neoplasia/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Autorrenovación de las Células/fisiología , Glioblastoma/patología , Humanos , Ratones , Células Madre Neoplásicas/metabolismo

RESUMEN

Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.

Asunto(s)

Glioblastoma/genética , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos

RESUMEN

The cranial window (CW) technique provides a simple and low-cost method to assess tumor angiogenesis in the brain. The CW combined with histology using selective markers for tumor and endothelial cells can allow a sensitive monitoring of novel antiangiogenesis therapies in preclinical models. The CW was established in cyclosporine immunosuppressed rats that were stereotactically grafted with fluorescent U87MG glioblastoma cells. One to 3 weeks after grafting, brain vasculature was visualized in vivo and assessed by immunofluorescence microscopy using antibodies against endothelial and smooth-muscle cells and blood brain barrier. At 1-2 weeks after grafting, the CW reliably detected the hypertrophy of venous-venous anastomoses and cortical veins. These structures increased highly significantly their pregrafting diameter. Arterialized veins and hemorrhages were seen by three weeks after grafting. Immunofluorescence microscopy showed significant branching and dilation of microvessels, particularly those surrounded by tumor cells. Mechanistically, these changes lead to loss of vascular resistance, increased venous outflow, and opening of venous-venous anastomoses on the cortical surface. Data from the present study, namely, the hypertrophy of cortical venous-venous anastomoses, microvessel branching, and dilation of the microvessels surrounded by tumor cells, indicate the power of this in vivo model for the sensitive monitoring of early tumor angiogenesis.

Asunto(s)

Bioensayo , Neoplasias Encefálicas , Encéfalo , Venas Cerebrales , Glioblastoma , Neoplasias Experimentales , Neovascularización Patológica , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Venas Cerebrales/metabolismo , Venas Cerebrales/patología , Glioblastoma/irrigación sanguínea , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Ratas , Ratas Wistar

RESUMEN

Abnormalities in arterial versus venous endothelial cell identity and dysregulation of angiogenesis are deemed important in the pathophysiology of brain arteriovenous malformations (AVMs). The Sonic hedgehog (Shh) pathway is crucial for both angiogenesis and arterial versus venous differentiation of endothelial cells, through its dual role on the vascular endothelial growth factor/Notch signaling and the nuclear orphan receptor COUP-TFII. In this study, we show that Shh, Gli1 (the main transcription factor of the Shh pathway), and COUP-TFII (a target of the non-canonical Shh pathway) are aberrantly expressed in human brain AVMs. We also show that implantation of pellets containing Shh in the cornea of Efnb2/LacZ mice induces growth of distinct arteries and veins, interconnected by complex sets of arteriovenous shunts, without an interposed capillary bed, as seen in AVMs. We also demonstrate that injection in the rat brain of a plasmid containing the human Shh gene induces the growth of tangles of tortuous and dilated vessels, in part positive and in part negative for the arterial marker αSMA, with direct connections between αSMA-positive and -negative vessels. In summary, we show that the Shh pathway is active in human brain AVMs and that Shh-induced angiogenesis has characteristics reminiscent of those seen in AVMs in humans.

Asunto(s)

Malformaciones Arteriovenosas/metabolismo , Encéfalo/fisiopatología , Proteínas Hedgehog/metabolismo , Animales , Humanos

RESUMEN

The question whether perivascular glioma cells invading the brain far from the tumor bulk may disrupt the blood-brain barrier (BBB) represents a crucial issue because under this condition tumor cells would be no more protected from the reach of chemotherapeutic drugs. A recent in vivo study that used human xenolines, demonstrated that single glioma cells migrating away from the tumor bulk are sufficient to breach the BBB. Here, we used brain xenografts of patient-derived glioma stem-like cells (GSCs) to show by immunostaining that in spite of massive perivascular invasion, BBB integrity was preserved in the majority of vessels located outside the tumor bulk. Interestingly, the tumor cells that invaded the brain for the longest distances traveled along vessels with retained BBB integrity. In surgical specimens of malignant glioma, the area of brain invasion showed several vessels with preserved BBB that were surrounded by tumor cells. On transmission electron microscopy, the cell inter-junctions and basal lamina of the brain endothelium were preserved even in conditions in which the tumor cells lay adjacently to blood vessels. In conclusion, BBB integrity associates with extensive perivascular invasion of glioma cells.

RESUMEN

Inhibitors of Vascular Endothelial Growth Factor target both tumor vasculature and cancer cells that have hijacked VEGF Receptors (VEGFRs) signaling for tumor growth-promoting activities. It is important to get precise insight in the specificity of cell responses to these antiangiogenic drugs to maximize their efficiency and minimize off-target systemic toxicity. Here we report that Axitinib, an inhibitor of VEGFRs currently in use as a second line treatment for advanced renal cell carcinoma, promotes senescence of human endothelial cells in vitro. A one-hour pulse of Axitinib is sufficient for triggering cell senescence. Mechanistically, this requires oxidative stress-dependent activation of the Ataxia Telangiectasia Mutated (ATM) kinase. Axitinib-mediated senescence promoting action is prevented by short-term treatment with antioxidants or ATM inhibitors, which conversely fail to prevent senescence induced by the DNA-damaging drug doxorubicin. Coherently, induction of oxidative stress-related genes distinguishes the response of endothelial cells to Axitinib from that to doxorubicin. Importantly, an Axitinib pulse causes cell senescence in glioblastoma cells. However, neither antioxidants nor ATM inhibitors can reverse this phenotype. Thus, antioxidants may selectively protect endothelial cells from Axitinib by decreasing systemic toxicity and maintaining a functional vascularization necessary for efficient delivery of chemotherapeutic drugs within the tumor mass.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Axitinib/farmacología , Senescencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Antioxidantes/farmacología , Células Endoteliales/metabolismo , Activación Enzimática , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Patológica/prevención & control , Inhibidores de Proteínas Quinasas/administración & dosificación

RESUMEN

Bevacizumab, a VEGF-targeting monoclonal antibody, may trigger an infiltrative growth pattern in glioblastoma. We investigated this pattern using both a human specimen and rat models. In the human specimen, a substantial fraction of infiltrating tumor cells were located along perivascular spaces in close relationship with endothelial cells. Brain xenografts of U87MG cells treated with bevacizumab were smaller than controls (p = 0.0055; Student t-test), however, bands of tumor cells spread through the brain farther than controls (p < 0.001; Student t-test). Infiltrating tumor Cells exhibited tropism for vascular structures and propensity to form tubules and niches with endothelial cells. Molecularly, bevacizumab triggered an epithelial to mesenchymal transition with over-expression of the receptor Plexin Domain Containing 1 (PLXDC1). These results were validated using brain xenografts of patient-derived glioma stem-like cells. Enforced expression of PLXDC1 in U87MG cells promoted brain infiltration along perivascular spaces. Importantly, PLXDC1 inhibition prevented perivascular infiltration and significantly increased the survival of bevacizumab-treated rats. Our study indicates that bevacizumab-induced brain infiltration is driven by vascular endothelium and depends on PLXDC1 activation of tumor cells.

Asunto(s)

Antineoplásicos Inmunológicos/farmacología , Bevacizumab/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Endotelio/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Adulto , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Bevacizumab/uso terapéutico , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Resistencia a Antineoplásicos , Células Endoteliales , Endotelio/citología , Endotelio/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Masculino , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Desnudas , Receptores de Superficie Celular/genética , Análisis de Supervivencia , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The role of autophagy in cancer onset and progression appears still controversial. On one hand, autophagy allows cancer cell to survive in unfavorable environmental conditions, on the other hand, once internal energy resources are exhausted, it leads to cell death. In addition, autophagy interpheres with cell cycle progression, de facto exerting a cytostatic activity. Hence, it represents an important target for anticancer therapy. For example, temozolomide (TMZ), of use for glioblastoma (GBM) treatment, appears as capable of inducing autophagy partially inhibiting cancer cell proliferation. However, GBM, a very aggressive brain tumor with poor prognosis even after surgery and radio-chemotherapy, invariably recurs and leads to patient death. Since cancer stem cells have been hypothesized to play a role in refractory/relapsing cancers, in the present work we investigated if autophagy could represent a constitutive cytoprotection mechanism for glioblastoma stem-like cells (GSCs) and if the modulation of autophagic process could affect GBM growth and survival. Thus, in the present study we first evaluated the relevance of autophagy in GBM tumor specimens, then its occurrence in GSCs and, finally, if modulation of autophagy could influence GSC response to TMZ. Our results suggested that, in vitro, the impairing autophagic process with quinacrine, a compound able to cross the blood-brain barrier, increased GSC susceptibility to TMZ. Death of GSCs was apparently due to the iron dependent form of programmed cell death characterized by the accumulation of lipid peroxides called ferroptosis. These results underscore the relevance of the modulation of autophagy in the GSC survival and death and suggest that triggering of ferroptosis in GSCs could represent a novel and important target for the management of glioblastoma.

Asunto(s)

Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias Encefálicas/patología , Glioblastoma/patología , Temozolomida/farmacología , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Asociadas a Microtúbulos/metabolismo , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Quinacrina/farmacología , Quinacrina/uso terapéutico , Proteína Sequestosoma-1/metabolismo , Temozolomida/uso terapéutico , Trasplante Heterólogo

RESUMEN

We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.

Asunto(s)

Neoplasias Encefálicas/patología , Transdiferenciación Celular/fisiología , Células Endoteliales/patología , Glioblastoma/patología , Recurrencia Local de Neoplasia/patología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/radioterapia , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Humanos , Hibridación Fluorescente in Situ , Ratones , Recurrencia Local de Neoplasia/genética

RESUMEN

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) represent an attractive tool for cell-based cancer therapy mainly because of their ability to migrate to tumors and to release bioactive molecules. However, the impact of MSCs on tumor growth has not been fully established. We previously demonstrated that murine MSCs show a strong tropism towards glioblastoma (GBM) brain xenografts and that these cells are able to uptake and release the chemotherapeutic drug paclitaxel (PTX), maintaining their tropism towards the tumor. Here, we address the therapy-relevant issue of using MSCs from human donors (hMSCs) for local or systemic administration in orthotopic GBM models, including xenografts of patient-derived glioma stem cells (GSCs). METHODS: U87MG or GSC1 cells expressing the green fluorescent protein (GFP) were grafted onto the striatum of immunosuppressed rats. Adipose hMSCs (Ad-hMSCs), fluorescently labeled with the mCherry protein, were inoculated adjacent to or into the tumor. In rats bearing U87MG xenografts, systemic injections of Ad-hMSCs or bone marrow (BM)-hMSCs were done via the femoral vein or carotid artery. In each experiment, either PTX-loaded or unloaded hMSCs were used. To characterize the effects of hMSCs on tumor growth, we analyzed survival, tumor volume, tumor cell proliferation, and microvascular density. RESULTS: Overall, the AD-hMSCs showed remarkable tropism towards the tumor. Intracerebral injection of Ad-hMSCs significantly improved the survival of rats with U87MG xenografts. This effect was associated with a reduction in tumor growth, tumor cell proliferation, and microvascular density. In GSC1 xenografts, intratumoral injection of Ad-hMSCs depleted the tumor cell population and induced migration of resident microglial cells. Overall, PTX loading did not significantly enhance the antitumor potential of hMSCs. Systemically injected Ad- and BM-hMSCs homed to tumor xenografts. The efficiency of hMSC homing ranged between 0.02 and 0.5% of the injected cells, depending both on the route of cell injection and on the source from which the hMSCs were derived. Importantly, systemically injected PTX-loaded hMSCs that homed to the xenograft induced cytotoxic damage to the surrounding tumor cells. CONCLUSIONS: hMSCs have a therapeutic potential in GBM brain xenografts which is also expressed against the GSC population. In this context, PTX loading of hMSCs seems to play a minor role.

Asunto(s)

Proliferación Celular , Glioblastoma/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Línea Celular Tumoral , Terapia Combinada , Glioblastoma/patología , Humanos , Ratones , Paclitaxel/administración & dosificación , Ratas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

INTRODUCTION: The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment. METHODS: U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage. RESULTS: Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor. CONCLUSIONS: MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.

Asunto(s)

Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Trasplante de Células Madre Mesenquimatosas , Paclitaxel/administración & dosificación , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Antineoplásicos Fitogénicos/toxicidad , Línea Celular Tumoral , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Paclitaxel/uso terapéutico , Paclitaxel/toxicidad , Ratas , Ratas Wistar

RESUMEN

In the neocortex, the coexistence of temporally locked excitation and inhibition governs complex network activity underlying cognitive functions, and is believed to be altered in several brain diseases. Here we show that this equilibrium can be unlocked by increased activity of layer 5 pyramidal neurons of the mouse neocortex. Somatic depolarization or short bursts of action potentials of layer 5 pyramidal neurons induced a selective long-term potentiation of GABAergic synapses (LTPi) without affecting glutamatergic inputs. Remarkably, LTPi was selective for perisomatic inhibition from parvalbumin basket cells, leaving dendritic inhibition intact. It relied on retrograde signaling of nitric oxide, which persistently altered presynaptic GABA release and diffused to inhibitory synapses impinging on adjacent pyramidal neurons. LTPi reduced the time window of synaptic summation and increased the temporal precision of spike generation. Thus, increases in single cortical pyramidal neuron activity can induce an interneuron-selective GABAergic plasticity effectively altering the computation of temporally coded information.

Asunto(s)

Células Piramidales/fisiología , Potenciales de Acción/fisiología , Animales , Canales de Calcio Tipo L/fisiología , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Ratones Endogámicos C57BL , Neocórtex/citología , Plasticidad Neuronal/fisiología , Neuronas , Técnicas de Placa-Clamp , Ácido gamma-Aminobutírico/fisiología

RESUMEN

SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.

Asunto(s)

Ácido Glutámico/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Transmisión Sináptica/fisiología , Proteína 25 Asociada a Sinaptosomas/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Humanos , Ratones , Plasticidad Neuronal/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/fisiología , Proteína 25 Asociada a Sinaptosomas/antagonistas & inhibidores , Proteína 25 Asociada a Sinaptosomas/genética , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

Asunto(s)

Neoplasias del Sistema Nervioso Central/patología , Proteínas de Unión al ADN/metabolismo , Glioblastoma/patología , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/metabolismo , Angiopoyetina 2/metabolismo , Animales , Biomarcadores/metabolismo , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/metabolismo , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Janus Quinasa 1/metabolismo , Janus Quinasa 1/farmacología , Ratones , Células Madre Neoplásicas/patología , Fosforilación , ARN Interferente Pequeño , Proteínas Represoras , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/genética , Células Tumorales Cultivadas

RESUMEN

The adult hippocampus is involved in learning and memory. As a consequence, it is a brain region of remarkable plasticity. This plasticity exhibits itself both as cellular changes and neurogenesis. For neurogenesis to occur, a population of local stem cells and progenitor cells is maintained in the adult brain and these are able to proliferate and differentiate into neurons which contribute to the hippocampal circuitry. There is much interest in understanding the role of immature cells in the hippocampus, in relation to learning and memory. Methods and mechanisms that increase the numbers of these cells will be valuable in this research field. We show here that single injections of soluble factors into the lateral ventricle of adult rats and mice induces the rapid (within one week) increase in the number of putative stem cells/progenitor cells in the hippocampus. The established progenitor marker Sox2 together with the more recently established marker Hes3, were used to quantify the manipulation of the Sox2/Hes3 double-positive cell population. We report that in both adult rodent species, Sox2+/Hes3+ cell numbers can be increased within one week. The most prominent increase was observed in the hilus of the dentate gyrus. This study presents a fast, pharmacological method to manipulate the numbers of endogenous putative stem cells/progenitor cells. This method may be easily modified to alter the degree of activation (e.g. by the use of osmotic pumps for delivery, or by repeat injections through implanted cannulas), in order to be best adapted to different paradigms of research (neurodegenerative disease, neuroprotection, learning, memory, plasticity, etc).

Asunto(s)

Envejecimiento/metabolismo , Proteínas de Unión al ADN/metabolismo , Hipocampo/citología , Proteínas Recombinantes/farmacología , Factores de Transcripción SOXB1/metabolismo , Animales , Animales Recién Nacidos , Recuento de Células , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Hipocampo/anatomía & histología , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Solubilidad

RESUMEN

Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimer's disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimer's disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3-dependent mechanism that drives synaptic failure and contributes to cognitive dysfunction in Alzheimer's disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Caspasa 3/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Transmisión Sináptica/fisiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Calcineurina/metabolismo , Inhibidores de Caspasas , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Hipocampo/fisiopatología , Trastornos de la Memoria/genética , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Transgénicos , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Oligopéptidos/farmacología , Ácido Poliglutámico/farmacología , Receptores AMPA/metabolismo

RESUMEN

Control of pyramidal neuron excitability is vital for the functioning of the neocortex. Somatodendritic slow self-inhibition (SSI) allows inhibitory neurons to regulate their own activity, but the existence of similar mechanisms in excitatory cells has not been shown. We found that in rodents endocannabinoids mediated SSI and long-term modulation of inhibitory connections in layer 2/3 pyramidal neurons with a distinct dendritic morphology, suggesting that a glutamatergic network in cortical circuits is self-regulated.

Asunto(s)

Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Neocórtex/citología , Inhibición Neural/fisiología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Animales , Benzoxazinas/farmacología , Cannabinoides/farmacología , Dendritas/fisiología , Ácido Glutámico/fisiología , Ratones , Morfolinas/farmacología , Naftalenos/farmacología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Piramidales/ultraestructura , Ratas , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/fisiología , Ácido gamma-Aminobutírico/fisiología

RESUMEN

In the CNS, endocannabinoids are identified mainly as two endogenous lipids: anandamide, the ethanolamide of arachidonic acid, and 2-arachidonoylglycerol (2-AG). Endocannabinoids are known to inhibit transmitter release from presynaptic terminals; however we have recently demonstrated that they are also involved in slow self-inhibition (SSI) of layer V low-threshold spiking (LTS) interneurons in rat somatosensory cortex. SSI is induced by repetitive firing in LTS cells, which can express either cholecystokinin or somatostatin. SSI is triggered by an endocannabinoid-dependent activation of a prolonged somatodendritic K(+) conductance and associated hyperpolarization in the same cell. The synthesis of both endocannabinoids is dependent on elevated [Ca(2+)](i) such as occurs during sustained neuronal activity. To establish whether 2-AG mediates autocrine LTS-SSI, we blocked its biosynthesis from phospholipase C (PLC) and diacylglycerol lipases (DAGLs). Current-clamp recordings from LTS interneurons in acute neocortical slices showed that inclusion of DAGL inhibitors in the whole-cell pipette prevented the long-lasting hyperpolarization triggered by LTS cell repetitive firing. Similarly, extracellular applications of a PLC inhibitor prevented SSI in LTS interneurons. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the CB1 antagonist AM251, as well as by PLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than 2-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons.

Asunto(s)

Ácidos Araquidónicos/metabolismo , Glicéridos/metabolismo , Interneuronas/metabolismo , Neocórtex/metabolismo , Inhibición Neural/fisiología , Animales , Endocannabinoides , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Interneuronas/efectos de los fármacos , Lipoproteína Lipasa/antagonistas & inhibidores , Lipoproteína Lipasa/efectos de los fármacos , Neocórtex/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley

RESUMEN

Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3-4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits.

Asunto(s)

Diferenciación Celular/fisiología , Hipocampo/citología , Memoria , Plasticidad Neuronal/fisiología , Neuronas/citología , Animales , Genes Supresores de Tumor , Hipocampo/fisiología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Transgénicos , Modelos Animales , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/fisiología , Percepción Espacial/fisiología , Factores de Tiempo , Proteínas Supresoras de Tumor