Cerebrovascular pathology during the progression of experimental Alzheimer's disease.

Clinical and experimental evidence point to a possible role of cerebrovascular dysfunction in Alzheimer's disease (AD). The 5xFAD mouse model of AD expresses human amyloid precursor protein and presenilin genes with mutations found in AD patients. It remains unknown whether amyloid deposition driven by these mutations is associated with cerebrovascular changes. 5xFAD and wild type mice (2 to 12months old; M2 to M12) were used. Thinned skull in vivo 2-photon microscopy was used to determine Aß accumulation on leptomeningeal or superficial cortical vessels over time. Parenchymal microvascular damage was assessed using FITC-microangiography. Collagen-IV and CD31 were used to stain basal lamina and endothelial cells. Methoxy-XO4, Thioflavin-S or 6E10 were used to visualize Aß accumulation in living mice or in fixed brain tissues. Positioning of reactive IBA1 microglia and GFAP astrocytes at the vasculature was rendered using confocal microscopy. Platelet-derived growth factor receptor beta (PDGFRß) staining was used to visualize perivascular pericytes. In vivo 2-photon microscopy revealed Methoxy-XO4(+) amyloid perivascular deposits on leptomeningeal and penetrating cortical vessels in 5xFAD mice, typical of cerebral amyloid angiopathy (CAA). Amyloid deposits were visible in vivo at M3 and aggravated over time. Progressive microvascular damage was concomitant to parenchymal Aß plaque accumulation in 5xFAD mice. Microvascular inflammation in 5xFAD mice presented with sporadic FITC-albumin leakages at M4 becoming more prevalent at M9 and M12. 3D colocalization showed inflammatory IBA1(+) microglia proximal to microvascular FITC-albumin leaks. The number of perivascular PDGFRß(+) pericytes was significantly decreased at M4 in the fronto-parietal cortices, with a trend decrease observed in the other structures. At M9-M12, PDGFRß(+) pericytes displayed hypertrophic perivascular ramifications contiguous to reactive microglia. Cerebral amyloid angiopathy and microvascular inflammation occur in 5xFAD mice concomitantly to parenchymal plaque deposition. The prospect of cerebrovascular pharmacology in AD is discussed.

Redistribution of PDGFRß cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus.

PURPOSE: The role of cerebrovascular dysfunction in seizure disorders is recognized. Blood-brain barrier (BBB) damage in epilepsy has been linked to endothelial and glial pathophysiological changes. Little is known about the involvement of pericytes, a cell type that contributes to BBB function. METHODS: NG2DsRed mice were used to visualize cerebrovascular pericytes. The pattern of vascular and parenchymal distributions of platelet-derived growth factor receptor beta (PDGFRß) cells was evaluated by immunohistochemistry. Status epilepticus was induced in NG2DsRed or C57BL/6J mice by intraperitoneal kainic acid (KA). Animals were perfused intracardially using FITC-Dextran or FITC-Albumin to visualize the cerebrovasculature. Colocalization was performed between NG2DsRed, PDGFRß and microglia IBA-1. Confocal 3D vessel reconstruction was used to visualize changes in cell morphology and position. PDGFRß expression was also evaluated in vitro using organotypic hippocampal cultures (OHC) treated with kainic acid to induce seizure-like activity. Co-localization of PDGFRß with the vascular marker RECA-1 and NG2 was performed. Finally, we assessed the expression of PDGFRß in brain specimens obtained from a cohort of patients affected by drug resistant epilepsy compared to available autoptic brain. RESULTS: In vivo, severe status epilepticus (SE) altered NG2DsRed vascular coverage. We found dishomogenous NG2DsRed perivascular ramifications after SE and compared to control. Concomitantly, PDGFRß(+) cells re-distributed towards the cerebrovasculature after severe SE. Cerebrovascular NG2DsRed partially colocalized with PDGFRß(+) while parenchymal PDGFRß(+) cells did not colocalize with IBA-1(+) microglia. Using in vitro OHC we found decreased NG2 vascular staining and increased PDGFRß(+) ramifications associated with RECA-1(+) microvessels after seizure-like activity. Cellular PDGFRß and NG2(+) colocalization was observed in the parenchyma. Finally, analysis of human TLE brains revealed perivascular and parenchymal PDGFRß(+) cell distributions resembling the murine in vivo and in vitro results. PDGFRß(+) cells at the cerebrovasculature were more frequent in TLE brain tissues as compared to the autoptic control. CONCLUSIONS: The rearrangement of PDGFRß(+) and vascular NG2DsRed cells after SE suggests a possible involvement of pericytes in the cerebrovascular modifications observed in epilepsy. The functional role of vascular-parenchymal PDGFRß(+) cell redistribution and the relevance of a pericyte response to SE remain to be fully elucidated.

Epileptiform activity induces vascular remodeling and zonula occludens 1 downregulation in organotypic hippocampal cultures: role of VEGF signaling pathways.

Recent studies suggest that blood-brain barrier (BBB) permeability contributes to epileptogenesis in symptomatic epilepsies. We have previously described angiogenesis, aberrant vascularization, and BBB alteration in drug-refractory temporal lobe epilepsy. Here, we investigated the role of vascular endothelial growth factor (VEGF) in an in vitro integrative model of vascular remodeling induced by epileptiform activity in rat organotypic hippocampal cultures. After kainate-induced seizure-like events (SLEs), we observed an overexpression of VEGF and VEGF receptor-2 (VEGFR-2) as well as receptor activation. Vascular density and branching were significantly increased, whereas zonula occludens 1 (ZO-1), a key protein of tight junctions (TJs), was downregulated. These effects were fully prevented by VEGF neutralization. Using selective inhibitors of VEGFR-2 signaling pathways, we found that phosphatidylinositol 3-kinase is involved in cell survival, protein kinase C (PKC) in vascularization, and Src in ZO-1 regulation. Recombinant VEGF reproduced the kainate-induced vascular changes. As in the kainate model, VEGFR-2 and Src were involved in ZO-1 downregulation. These results showed that VEGF/VEGFR-2 initiates the vascular remodeling induced by SLEs and pointed out the roles of PKC in vascularization and Src in TJ dysfunction, respectively. This suggests that Src pathway could be a therapeutic target for BBB protection in epilepsies.

Extracellular interactions between GluR2 and N-cadherin in spine regulation.

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.

Angiogenesis is associated with blood-brain barrier permeability in temporal lobe epilepsy.

Previous studies from our group, focusing on neuro-glial remodelling in human temporal lobe epilepsy (TLE), have shown the presence of immature vascular cells in various areas of the hippocampus. Here, we investigated angiogenic processes in hippocampi surgically removed from adult patients suffering from chronic intractable TLE, with various aetiologies. We compared hippocampi from TLE patients to hippocampi obtained after surgery or autopsy from non-epileptic patients (NE). We quantified the vascular density, checked for the expression of angiogenic factors and their receptors and looked for any blood-brain barrier (BBB) leakage. We used a relevant model of rat limbic epilepsy, induced by lithium-pilocarpine treatment, to understand the sequence of events. In humans, the vessel density was significantly higher in TLE than in NE patients. This was neither dependent on the aetiology nor on the degree of neuronal loss, but was positively correlated with seizure frequency. In the whole hippocampus, we observed many complex, tortuous microvessels. In the dentate gyrus, when the granular layer was dispersed, long microvessels appeared radially orientated. Vascular endothelial factor (VEGF) and tyrosine kinase receptors were detected in different types of cells. An impairment of the BBB was demonstrated by the loss of tight junctions and by Immunoglobulines G (IgG) leakage and accumulation in neurons. In the rat model of TLE, VEGF over-expression and BBB impairment occurred early after status epilepticus, followed by a progressive increase in vascularization. In humans and rodents, angiogenic processes and BBB disruption were still obvious in the chronic focus, probably activated by recurrent seizures. We suggest that the persistent leakage of serum IgG in the interstitial space and their uptake by neurons may participate in hypoperfusion and in neuronal dysfunction occurring in TLE.

Lack of CAR impacts neuronal function and cerebrovascular integrity in vivo.

Nuclear receptors (NRs) are a group of transcription factors emerging as players in normal and pathological CNS development. Clinically, an association between the constitutive androstane NR (CAR) and cognitive impairment was proposed, however never experimentally investigated. We wished to test the hypothesis that the impact of CAR on neurophysiology and behavior is underlined by cerebrovascular-neuronal modifications. We have used CAR(-/-) C57BL/6 and wild type mice and performed a battery of behavioral tests (recognition, memory, motor coordination, learning and anxiety) as well as longitudinal video-electroencephalographic recordings (EEG). Brain cell morphology was assessed using 2-photon or electron microscopy and fluorescent immunohistochemistry. We observed recognition memory impairment and increased anxiety-like behavior in CAR(-/-) mice, while locomotor activity was not affected. Concomitantly to memory deficits, EEG monitoring revealed a decrease in 3.5-7Hz waves during the awake/exploration and sleep periods. Behavioral and EEG abnormalities in CAR(-/-) mice mirrored structural changes, including tortuous fronto-parietal penetrating vessels. At the cellular level we found reduced ZO-1, but not CLDN5, tight junction protein expression in cortical and hippocampal isolated microvessel preparations. Interestingly, the neurotoxin kainic acid, when injected peripherally, provoked a rapid onset of generalized convulsions in CAR(-/-) as compared to WT mice, supporting the hypothesis of vascular permeability. The morphological phenotype of CAR(-/-) mice also included some modifications of GFAP/IBA1 glial cells in the parenchymal or adjacent to collagen-IV(+) or FITC(+) microvessels. Neuronal defects were also observed including increased cortical NEUN(+) cell density, hippocampal granule cell dispersion and increased NPY immunoreactivity in the CA1 region in CAR(-/-) mice. The latter may contribute to the in vivo phenotype. Our results indicate that behavioral and electroencephalographic changes in adult CAR(-/-) mice are concomitant to discrete developmental or structural brain defects. The latter could increase the vulnerability to neurotoxins. The possibility that interfering with nuclear receptors during development could contribute to adulthood brain changes is proposed.

Inflammatory reactions in human medial temporal lobe epilepsy with hippocampal sclerosis.

Many experimental studies suggest that NFkappaB, a transcription factor involved in acute inflammation, and cytokines participate in neuronal excitability and/or glial scar formation in epilepsy. In this report, we looked for the expression of NFkappaB in hippocampi surgically removed in patients with medial temporal lobe epilepsy (MTLE) and hippocampal sclerosis (HS) who had an history of febrile convulsions. We analyzed 18 hippocampi from epileptic patients with MTLE and HS, and we used as control specimens three hippocampi from non-epileptic patients and four hippocampi from patients with cryptogenic MTLE without HS. We used antibodies raised against the NFkappaB-p65 subunit and we identified glial cells with specific antibodies. Hippocampi from patients with MTLE and HS displayed severe neuronal loss surrounded by gliosis in CA1 area and more or less in CA3/CA4 areas. Double immunolabeling showed that reactive astrocytes of lesioned areas over-expressed NFkappaB-p65 (significantly when compared to control specimens). Moreover, some surviving pyramidal neurons in these areas and numerous dentate granule cells were strongly positive for NFkappaB-p65 in cytoplasm and nucleus, whereas control hippocampi showed a faint basal cytoplasmic staining in neurons. These results suggest that in epileptic hippocampi with typical sclerosis, inflammatory processes are chronically active or transiently re-induced by recurrent seizures. Whether NFkappaB over-expression reflects protective or deleterious mechanisms in the epileptic focus remains to be elucidated.

Immature-like astrocytes are associated with dentate granule cell migration in human temporal lobe epilepsy.

In human temporal lobe epilepsy, a dispersion of dentate granule cells is frequently described in adults who had an early risk factor. To elucidate the role of glia in this phenomenon, we investigated neuronal dispersion, astrocyte organization and expression of intermediate filaments of mature and immature astrocytes (i.e. glial fibrillary acidic protein (GFAP) and vimentin, respectively) in seven subjects with early febrile seizures (F(+)) and five subjects with other etiologies than febrile seizures (F(-)). Compared to F(-) patients, a majority of F(+) subjects showed neuronal dispersion and vimentin expression in radial glia. However, in two patients with the maximal dispersion, radial processes expressed only GFAP. We suggest that granule cell migration that occurs in adult epileptic focus results from the transient occurrence of immature-like glia throughout the granular layer.

IgG leakage may contribute to neuronal dysfunction in drug-refractory epilepsies with blood-brain barrier disruption.

Focal epilepsies are often associated with blood-brain barrier disruption. In 4 entorhinal cortex tissue samples and 13 hippocampal samples from patients with pharmacoresistent temporal lobe epilepsy, we observed immunoglobulin G (IgG) leakage in the parenchyma and IgG-positive neurons that had evidence of neurodegeneration, such as shrinkage and eosinophilia. These findings were not present in samples from 12 nonepileptic control subjects. To complement these findings, we used a rat in vivo model that mimics the development of limbic epilepsy with blood-brain barrier disruption. During epileptogenesis, IgG leakage and neuronal IgG uptake increased concomitantly with the occurrence of seizures. Immunoglobulin G accumulation in neurons was selective, particularly for interneurons and pyramidal neurons. Immunohistochemistry and electron microscopy showed that IgG uptake in the rat neurons was associated with eosinophilia, shrinkage, and ultrastructural degenerative changes. Moreover, IgG-positive neurons lost their NeuN immunohistochemical staining. Together, these observations suggest that IgG leakage is related to neuronal impairment and may be a pathogenic mechanism in epileptogenesis and chronic epilepsy.

Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors.

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.

Increased number of neural progenitors in human temporal lobe epilepsy.

An increased neurogenesis is reported in animal models of mesial temporal lobe epilepsy (MTLE) but the fate of newborn cells is unknown. Here, we attempted to demonstrate neurogenesis in adult epileptic tissue obtained after hippocampectomy. MTLE hippocampi showed increased expression of division markers and of Musashi-1, a marker of neural progenitors, compared to control hippocampi. Large quantities of Musashi-1+ cells were obvious in the subgranular layer and the subventricular zone, both known neurogenic areas, and in the fissura hippocampi. Musashi-1 was expressed by small cells that were mainly vimentin+ or nestin+, rarely Dcx+ or PSA-NCAM+ and negative for markers of mature neurons or astrocytes. Some of them are present in the granular layer, the hilus, and CA1 area resembling the ectopic positions described in rodents. These findings demonstrate that neural progenitors proliferate in chronic epilepsy and suggest that the fissura hippocampi behaves like another neurogenic area.