Jorge Marcondes de Souza

Astrocyte-induced Synaptogenesis Is Mediated by Transforming Growth Factor β Signaling through Modulation of d-Serine Levels in Cerebral Cortex Neurons

Background: Synapse formation and function is modulated by intrinsic and extrinsic non-autonomous factors. Results: Astrocytes induce synapse formation through TGF-β1 pathway. TGF-β1 synaptogenic property is dependent on d-serine signaling. Conclusion: TGF-β induces excitatory glutamatergic synapses in vertebrates. Significance: This is a novel molecular mechanism that might impact synaptic function and shed light on new potential therapeutic targets for synaptic deficit diseases. Assembly of synapses requires proper coordination between pre- and postsynaptic elements. Identification of cellular and molecular events in synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for astrocytes in synapse formation and function has been proposed. Here, we show that transforming growth factor β (TGF-β) signaling is a novel synaptogenic pathway for cortical neurons induced by murine and human astrocytes. By combining gain and loss of function approaches, we show that TGF-β1 induces the formation of functional synapses in mice. Further, TGF-β1-induced synaptogenesis involves neuronal activity and secretion of the co-agonist of the NMDA receptor, d-serine. Manipulation of d-serine signaling, by either genetic or pharmacological inhibition, prevented the TGF-β1 synaptogenic effect. Our data show a novel molecular mechanism that might impact synaptic function and emphasize the evolutionary aspect of the synaptogenic property of astrocytes, thus shedding light on new potential therapeutic targets for synaptic deficit diseases.

Amyloid-β Oligomers Induce Differential Gene Expression in Adult Human Brain Slices

Background: Soluble Aβ oligomers (AβOs) have been increasingly proposed as the cause of synapse failure and cognitive dysfunction in Alzheimer disease. Results: Sublethal AβO concentrations induce changes in gene expression in adult human brain slices. Conclusion: AβOs impact transcription in important neuronal pathways preceding neurodegeneration. Significance: Results establish early mechanisms involved in AβO-triggered neuronal dysfunction in a novel human-derived experimental model. Cognitive decline in Alzheimer disease (AD) is increasingly attributed to the neuronal impact of soluble oligomers of the amyloid-β peptide (AβOs). Current knowledge on the molecular and cellular mechanisms underlying the toxicity of AβOs stems largely from rodent-derived cell/tissue culture experiments or from transgenic models of AD, which do not necessarily recapitulate the complexity of the human disease. Here, we used DNA microarray and RT-PCR to investigate changes in transcription in adult human cortical slices exposed to sublethal doses of AβOs. The results revealed a set of 27 genes that showed consistent differential expression upon exposure of slices from three different donors to AβOs. Functional classification of differentially expressed genes revealed that AβOs impact pathways important for neuronal physiology and known to be dysregulated in AD, including vesicle trafficking, cell adhesion, actin cytoskeleton dynamics, and insulin signaling. Most genes (70%) were down-regulated by AβO treatment, suggesting a predominantly inhibitory effect on the corresponding pathways. Significantly, AβOs induced down-regulation of synaptophysin, a presynaptic vesicle membrane protein, suggesting a mechanism by which oligomers cause synapse failure. The results provide insight into early mechanisms of pathogenesis of AD and suggest that the neuronal pathways affected by AβOs may be targets for the development of novel diagnostic or therapeutic approaches.

Single-center experience with the Neuroform stent for endovascular treatment of wide-necked intracranial aneurysms.

BACKGROUND Stent-assisted coiling is an accepted endovascular treatment (EVT) for wide-necked intracranial aneurysms. The Neuroform stent (Target Therapeutics, Fremont, Calif) is a flexible nitinol self-expandable stent that was designed to potentially overcome the limitations of balloon expandable coronary stents in the intracranial circulation. The aim of this study was to reenforce the use of this stent for EVT of wide-necked cerebral aneurysms. METHODS Between March 2005 and March 2008, 24 patients harboring wide-necked cerebral aneurysms were treated with stent reconstruction of the aneurysm neck. Inclusion criteria restricted the group to adult patients with wide-necked intracranial aneurysms (ruptured and unruptured lesions). Immediate postprocedure angiography studies were performed to determine successful coil occlusion of the aneurysm as well as patency of the parent vessel. We assessed the clinical history, aneurysm dimensions, and technical detail of the procedures, including any difficulties with stent placement and deployment, degree of aneurysm occlusion, and complications. Clinical outcome was assessed with the Glasgow Outcome Scale (GOS). RESULTS The stent was easily navigated and precisely positioned in 24 of 26 cases. However, technical difficulties occurred in 9 patients, including difficulties in crossing the stents interstice in 6 cases, inadvertent stent delivery (n = 1), and incapacity of stent delivery (n = 1) and incapacity of crossing the neck (n = 1). These latter 2 cases were classified as failures of the stent-assisted technique. A single procedural complication occurred, involving transient nonocclusive intrastent thrombus formation, which was treated uneventfully with abciximab. Seventeen patients experienced excellent clinical outcomes (GOS 5), with good outcomes (GOS 4) in 5 patients and a poor outcome (GOS 3) in 2 patients. There were no treatment-related deaths or neurologic complications (mean clinical follow-up, 12 months). Angiographic results consisted of 17 complete occlusions, 4 neck remnants, and 3 incomplete occlusions. CONCLUSIONS The Neuroform stent is very useful for EVT of wide-necked intracranial aneurysms because it is easy to navigate and to deploy accurately. In most cases, the stent can be deployed precisely, even in very tortuous carotid siphons. Although in some cases delivery and deployment was challenging, clinically significant complications were not observed.

Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes‐derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte‐induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF‐β1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF‐β pathway. TGF‐β1‐induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF‐β1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF‐β1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF‐β1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. GLIA 2014;62:1917–1931

Pituitary tuberculoma: an unusual lesion of sellar region.

Pituitary tuberculomas are extremely rare lesions, with only few cases described in the literature, usually mistaken as pituitary tumors. Its heterogeneous clinical and imaging profile preclude preoperative diagnosis which ultimately relies on the histopathological examination. We describe a 46 years old woman who presented with an episode of confusion and hypopituitarism with no evidence of systemic tuberculosis. Computed tomography (CT) showed a central calcified and enhancing sellar mass. Magnetic resonance imaging (MRI) showed a sellar mass with suprasellar extension and associated optic chiasm compression. She was submitted a craniotomy for biopsy and resection. A caseous material was found at the center of the lesion involved by a thick wall. Due to the wall adherence to the optic chiasm and the inflammatory aspects of the lesion, subtotal removal was achieved and the patient followed on anti-tuberculous and hormonal replacement therapy. Sellar tuberculomas should be considered in the differential diagnosis of sellar tumors in order to offer appropriate treatment.

Equinatoxin II Potentiates Temozolomide- and Etoposide-Induced Glioblastoma Cell Death

Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.

Cavernous carotid artery pseudo-aneurysm treated by stenting in acromegalic patient

We report on a case of endovascular management of pseudoaneurysm of the cavernous segment of the internal carotid artery with covered stent reconstruction. A 36 years-old woman with a history of previous transsphenoidal approach for pituitary macroadenoma and false aneurysma formation was studied in a protocol that included balloon test occlusion and cerebral blood flow evaluation. An endovascular covered stent deployment in the area of the carotid laceration was performed with isolation of the aneurysm from the circulation and maintenance of the carotid flow. Helical angio-CT and cerebral digital subtraction angiography showed the carotid preservation without stenosis in the stented area. In conclusion, endovascular stent reconstruction for post-transsphenoidal carotid artery laceration and false aneurysm is demonstrated as useful technical adjunct in the management strategy and with the potential for carotid sacrifice morbidity avoidance.

Connective Tissue Growth Factor (CTGF/CCN2) Is Negatively Regulated during Neuron-Glioblastoma Interaction

Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFβ luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.

Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against Aβ Oligomers in Alzheimer's Disease Model

Alzheimers disease (AD) is characterized by progressive cognitive decline, increasingly attributed to neuronal dysfunction induced by amyloid-β oligomers (AβOs). Although the impact of AβOs on neurons has been extensively studied, only recently have the possible effects of AβOs on astrocytes begun to be investigated. Given the key roles of astrocytes in synapse formation, plasticity, and function, we sought to investigate the impact of AβOs on astrocytes, and to determine whether this impact is related to the deleterious actions of AβOs on synapses. We found that AβOs interact with astrocytes, cause astrocyte activation and trigger abnormal generation of reactive oxygen species, which is accompanied by impairment of astrocyte neuroprotective potential in vitro. We further show that both murine and human astrocyte conditioned media (CM) increase synapse density, reduce AβOs binding, and prevent AβO-induced synapse loss in cultured hippocampal neurons. Both a neutralizing anti-transforming growth factor-β1 (TGF-β1) antibody and siRNA-mediated knockdown of TGF-β1, previously identified as an important synaptogenic factor secreted by astrocytes, abrogated the protective action of astrocyte CM against AβO-induced synapse loss. Notably, TGF-β1 prevented hippocampal dendritic spine loss and memory impairment in mice that received an intracerebroventricular infusion of AβOs. Results suggest that astrocyte-derived TGF-β1 is part of an endogenous mechanism that protects synapses against AβOs. By demonstrating that AβOs decrease astrocyte ability to protect synapses, our results unravel a new mechanism underlying the synaptotoxic action of AβOs in AD. SIGNIFICANCE STATEMENT Alzheimers disease is characterized by progressive cognitive decline, mainly attributed to synaptotoxicity of the amyloid-β oligomers (AβOs). Here, we investigated the impact of AβOs in astrocytes, a less known subject. We show that astrocytes prevent synapse loss induced by AβOs, via production of transforming growth factor-β1 (TGF-β1). We found that AβOs trigger morphological and functional alterations in astrocytes, and impair their neuroprotective potential. Notably, TGF-β1 reduced hippocampal dendritic spine loss and memory impairment in mice that received intracerebroventricular infusions of AβOs. Our results describe a new mechanism underlying the toxicity of AβOs and indicate novel therapeutic targets for Alzheimers disease, mainly focused on TGF-β1 and astrocytes.

Interaction of amyloid-β (Aβ) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice

Brain accumulation of the amyloid-β protein (Aβ) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aβ oligomers (AβOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AβOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AβOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AβOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AβO binding to hippocampal neurons and prevented AβO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AβOs in mice. The results indicate that Nrx2α and NL1 are targets of AβOs and that prevention of this interaction reduces the deleterious impact of AβOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AβOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.