Beatrice Paradiso

Brain-Derived Neurotrophic Factor mRNA and Protein Are Targeted to Discrete Dendritic Laminas by Events That Trigger Epileptogenesis

Dendritic targeting of mRNA and local protein synthesis are mechanisms that enable neurons to deliver proteins to specific postsynaptic sites. Here, we demonstrate that epileptogenic stimuli induce a dramatic accumulation of BDNF mRNA and protein in the dendrites of hippocampal neurons in vivo. BDNF mRNA and protein accumulate in dendrites in all hippocampal subfields after pilocarpine seizures and in selected subfields after other epileptogenic stimuli (kainate and kindling). BDNF accumulates selectively in discrete dendritic laminas, suggesting targeting to synapses that are active during seizures. Dendritic targeting of BDNF mRNA occurs during the time when the cellular changes that underlie epilepsy are occurring and is not seen after intense stimuli that are non-epileptogenic, including electroconvulsive seizures and high-frequency stimulation. MK801, an NMDA receptor antagonist that can prevent epileptogenesis but not acute seizures, prevents the dendritic accumulation of BDNF mRNA, indicating that dendritic targeting is mediated via NMDA receptor activation. Together, these results suggest that dendritic accumulation of BDNF mRNA and protein play a critical role in the cellular changes leading to epilepsy.

Localized delivery of fibroblast growth factor–2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor–2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures.

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1β expression. The effect appeared to be most prominent on IL-1β, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1β in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process.

Localized overexpression of FGF‐2 and BDNF in hippocampus reduces mossy fiber sprouting and spontaneous seizures up to 4 weeks after pilocarpine‐induced status epilepticus

Purpose:  We have recently reported that viral vector–mediated supplementation of fibroblast growth factor‐2 (FGF‐2) and brain‐derived neurotrophic factor (BDNF) in a lesioned, epileptogenic rat hippocampus limits neuronal damage, favors neurogenesis, and reduces spontaneous recurrent seizures. To test if this treatment can also prevent hippocampal circuit reorganization, we examined here its effect on mossy fiber sprouting, the best studied form of axonal plasticity in epilepsy.

A pathogenetic hypothesis of Unverricht-Lundborg disease onset and progression

Unverricht-Lundborg disease (EPM1), the most common progressive myoclonic epilepsy, is associated with a defect of cystatin B (CSTB), a protease inhibitor. We used CSTB knockout mice to test the hypothesis that EPM1 onset is related to a latent hyperexcitability and that progression depends on higher susceptibility to seizure-induced cell damage. Hippocampal slices prepared from CSTB-deficient mice were hyperexcitable, as they responded to afferent stimuli in CA1 with multiple population spikes and kainate perfusion provoked the appearance of epileptic-like activity earlier than in WT mice. This hyperexcitability may depend on loss of inhibition, because the density of GABA-immunoreactive cells was reduced in the hippocampus of CSTB knockouts. In vivo, CSTB-deficient mice treated with kainate displayed increased susceptibility to seizures, with shorter latency to seizure onset and increased seizure severity compared with WT littermates. Furthermore, a greater degree of neuronal damage was observed in CSTB-deficient than in WT mice after seizures of identical grade, indicating increased susceptibility to seizure-induced cell death.

Evaluation of cell damage in organotypic hippocampal slice culture from adult mouse: A potential model system to study neuroprotection

The use of organotypic hippocampal slice culture (OHSC) has become a powerful tool for studying cell damage in different neuropathological states, since it reproduces the basic morphological and functional properties of hippocampal neuronal network. However, the conventional OHSCs are established from postnatal animals rather than adult. Here we reevaluated the features of cell death in adult OHSC in detail and found potential utility for the study of neuroprotection. Organotypic culture of hippocampal slices from adult mice under conventional conditions led to a time-dependent and reproducible cell death. Around 6days in vitro (DIV), slices lost 50% of the cells, based on LDH release assessment. The cell death was greater than 90% after DIV 15. The cell loss was linearly correlated (r=0.944, P<0.01) with the time in culture. The electrophysiological responses to the stimulus in the cultured adult slices were accordingly reduced. The cell degeneration during adult OHSC might be utilized as a tool for studying neuroprotective effects in drug development. To illustrate this potential use, adult OHSCs were challenged with brain-derived neurotrophic factor (BDNF). We found that the continuous supplementation of 300ng/ml BDNF promoted cell survival of adult OHSC. Using immunohistochemistry and Western blot analyses of neuronal markers, we also demonstrated the pro-survival effects of BDNF on neurons in the adult OHSC system. It is suggested that OHSCs from adult mice might provide an alternative model system for neuronal degeneration, suitable for studying physiological factors and pharmacological compounds contributing to neuronal survival.

FGF-2 Overexpression Increases Excitability and Seizure Susceptibility but Decreases Seizure-Induced Cell Loss

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

Loss of cortical GABA terminals in Unverricht-Lundborg disease.

Unverricht-Lundborg disease (ULD) is the most common progressive myoclonic epilepsy. Its etiology has been identified in a defect of a protease inhibitor, cystatin B (CSTB), but the mechanism(s) by which this defect translates in the clinical manifestations of the disease are still obscure. We tested the hypothesis that ULD is accompanied by a loss of cortical GABA inhibition in a murine model (the CSTB knockout mouse) and in a human case. Cortical GABA signaling has been investigated measuring VGAT immunohistochemistry (a histological marker of the density of GABA terminals), GABA release from synaptosomes and paired-pulse stimulation. In CSTB knockout mice, a progressive decrease in neocortex thickness was found, associated with a prevalent loss of GABA interneurons. A marked reduction in VGAT labeling was found in the cortex of both CSTB knockout mice and an ULD patient. This implicates a reduction in GABA synaptic transmission, which was confirmed in the mouse model as reduction in GABA release from isolated nerve terminals and as loss of electrophysiologically measured GABA inhibition. The alterations in VGAT immunolabeling progressed in time, paralleling the worsening of myoclonus. These results provide direct evidence that loss of cortical GABA input occurs in a relevant animal model and in a case of human ULD, leading to a condition of latent hyperexcitability that favors myoclonus and seizures. These findings contribute to the understanding of the pathogenic mechanism of ULD and of the neurobiological basis of the effect of currently employed drugs.

Effects of defective herpes simplex vectors expressing neurotrophic factors on the proliferation and differentiation of nervous cells in vivo

Neurotrophic factors (NTFs) are known to govern the processes involved in central nervous system cell proliferation and differentiation. Thus, they represent very attractive candidates for use in the study and therapy of neurological disorders. We constructed recombinant herpesvirus-based-vectors capable of expressing fibroblast growth factor-2 (FGF-2) and ciliary neurotrophic factor (CNTF) alone or in combinations. In vitro, vectors expressing FGF-2 and CNTF together, but not those expressing either NTF alone, caused proliferation of O-2A progenitors. Furthermore, based on double-labeling experiments performed using markers for neurons (MAP-2), oligodendrocytes (CNPase) and astrocytes (GFAP), most of the new cells were identified as astrocytes, but many expressed neuronal or oligodendrocytic markers. In vivo, vectors have been injected in the rat hippocampus. At 1 month after inoculation, a highly significant increase in BrdU-positive cells was observed in the dentate gyrus of animals injected with the vector expressing FGF-2 and CNTF together, but not in those injected with vectors expressing the single NTFs. Furthermore, double-labeling experiments confirmed in vitro data, that is, most of the new cells identified as astrocytes, some as neurons or oligodendrocytes. These data show the feasibility of the vector approach to induce proliferation and differentiation of neurons and/or oligodendrocytes in vivo.