Early Chronic Carbamazepine-in-Food Administration to MAM/Pilocarpine Rats Does Not Affect Convulsive Motor Seizures.

Antiepileptic drug-resistance is a major health problem in patients with cortical dysplasia (CD). Whether drug-resistant epilepsy is associated with progressive brain damage is still debated. We previously generated a rat model of acquired CD, the methylazoxymethanol-pilocarpine (MP) rat, in which the occurrence of status epilepticus and subsequent spontaneous seizures induce progressive brain damage (Nobili et al., 2015). The present study tested the outcome of early-chronic carbamazepine (CBZ) administration on both seizure activity and brain damage in MP rats. We took advantage of the non-invasive CBZ-in-food administration protocol, established by Ali (2012), which proved effective in suppressing generalized convulsive seizures in kainic acid rat model of epilepsy. MP rats were treated immediately after the onset of the first spontaneous seizure with 300 mg/kg/day CBZ formulated in pellets for a two-months-trial. CBZ-treated rats were continuously video-monitored to detect seizure activity and were compared with untreated epileptic MP rats. Despite CBZ serum levels in treated rats were within the suggested therapeutic range for humans, CBZ affected spontaneous convulsive seizures in 2 out of 10 treated rats (responders), whereas the remaining animals (non-responders) did not show any difference when compared to untreated MP rats. Histological analysis revealed cortical thinning paralleled by robust staining of Fluoro-Jade+ (FJ+) degenerating neurons and diffuse tissue necrosis in CBZ-non-responder vs CBZ-responder rats. Data reported here suggest that MP rat model represents suitable experimental setting where to investigate mechanisms of CD-related drug-resistant epilepsy and to verify if modulation of seizures, with appropriate treatment, may reduce seizure-induced brain damage.

Targeting PSD95-nNOS interaction by Tat-N-dimer peptide during status epilepticus is neuroprotective in MAM-pilocarpine rat model.

Glutamate receptors play a crucial pathogenic role in brain damage induced by status epilepticus (SE). SE may initiate NMDAR-dependent excitotoxicity through the production of oxidative damage mediated by the activation of a ternary complex formed by the NMDA receptor, the post-synaptic density scaffolding protein 95 (PSD95) and the neuronal NO synthase (nNOS). The inhibition of the protein-protein-interaction (PPI) of the NMDAR-PSD95-nNOS complex is one of the most intriguing challenges recently developed to reduce neuronal death in both animal models and in patients with cerebral ischemia. We took advantage of this promising approach to verify whether early administration of a neuroprotective NMDAR-PSD95-nNOS PPI inhibitor preserves the brain from SE-induced damage in a model of acquired cortical dysplasia, the methylazoxymethanol (MAM)/pilocarpine rat. Pilocarpine-induced SE rapidly determined neurodegenerative changes mediated by a NMDAR-downstream neurotoxic pathway in MAM rats. We demonstrated that SE rapidly induces NMDAR activation, nNOS membrane translocation, PSD95-nNOS molecular interaction associated with neuronal and glial peroxynitrite accumulation in the neocortex of MAM-pilocarpine rats. These changes were paralleled by rapid c-fos overexpression and by progressive spectrin proteolysis, suggestive of calpain activity and irreversible cytoskeletal damage. Early administration of a cell-penetrating Tat-N-dimer peptide inhibitor of NMDAR-PSD95-nNOS PPI during SE significantly rescued the MAM-pilocarpine rats from SE-induced mortality, reduced the number of degenerating neurons, decreased neuronal c-fos activation, peroxynitrite formation and cytoskeletal degradation and prevented astrogliosis. Our findings suggest an overall neuroprotective effect of blocking PSD95-nNOS protein-protein-interaction against SE insult.

Axon outgrowth and neuronal differentiation defects after a-SMN and FL-SMN silencing in primary hippocampal cultures.

Spinal Muscular Atrophy (SMA) is a severe autosomal recessive disease characterized by selective motor neuron degeneration, caused by disruptions of the Survival of Motor Neuron 1 (Smn1) gene. The main product of SMN1 is the full-length SMN protein (FL-SMN), that plays an established role in mRNA splicing. FL-SMN is also involved in neurite outgrowth and axonal transport. A shorter SMN isoform, axonal-SMN or a-SMN, displays a more specific axonal localization and has remarkable axonogenic properties in NSC-34. Introduction of known SMA mutations into the a-SMN transcript leads to impairment of axon growth and morphological defects similar to those observed in SMA patients and animal models. Although there is increasing evidence for the relevance of SMN axonal functions in SMA pathogenesis, the specific contributions of FL-SMN and a-SMN are not known yet. This work aimed to analyze the differential roles of FL-SMN and a-SMN in axon outgrowth and in neuronal homeostasis during differentiation of neurons into a mature phenotype. We employed primary cultures of hippocampal neurons as a well-defined model of polarization and differentiation. By analyzing subcellular localization, we showed that a-SMN is preferentially localized in the growing axonal compartment. By specifically silencing FL-SMN or a-SMN proteins, we demonstrated that both proteins play a role in axon growth, as their selective down-regulation reduces axon length without affecting dendritic arborization. a-SMN silencing, and in minor extent FL-SMN silencing, resulted in the growth of multi-neuritic neurons, impaired in the differentiation process of selecting a single axon out of multiple neurites. In these neurons, neurites often display mixed axonal and dendritic markers and abnormal distribution of the axonal initial segment protein Ankirin G, suggesting loss of neuronal polarity. Our results indicate that a-SMN and FL-SMN are needed for neuronal polarization and organization of axonal and dendritic compartments, processes that are fundamental for neuronal function and survival.

Continuous neurodegeneration and death pathway activation in neurons and glia in an experimental model of severe chronic epilepsy.

Whether seizures might determine the activation of cell death pathways and what could be the relevance of seizure-induced cell death in epilepsy are still highly debated issues. We recently developed an experimental model of acquired focal cortical dysplasia (the MAM-pilocarpine or MP rat) in which the occurrence of status epilepticus--SE--and subsequent seizures induced progressive cellular/molecular abnormalities and neocortical/hippocampal atrophy. Here, we exploited the same model to verify when, where, and how cell death occurred in neurons and glia during epilepsy course. We analyzed Fluoro Jade (FJ) staining and the activation of c-Jun- and caspase-3-dependent pathways during epilepsy, from few hours post-SE up to six months of spontaneous recurrent seizures. FJ staining revealed that cell injury in MP rats was not temporally restricted to SE, but extended throughout the different epileptic stages. The region-specific pattern of FJ staining changed during epilepsy, and FJ(+) neurons became more prominent in the dorsal and ventral hippocampal CA at chronic epilepsy stages. Phospho-c-Jun- and caspase-3-dependent pathways were selectively activated respectively in neurons and glia, at early but even more conspicuously at late chronic stages. Phospho-c-Jun activation was associated with increased cytochrome-c staining, particularly at chronic stages, and the staining pattern of cytochrome-c was suggestive of its release from the mitochondria. Taken together, these data support the content that at least in the MP rat model the recurrence of seizures can also sustain cell death mechanisms, thus continuously contributing to the pathologic process triggered by the occurrence of SE.

Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms.

The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN.

Progressive brain damage, synaptic reorganization and NMDA activation in a model of epileptogenic cortical dysplasia.

Whether severe epilepsy could be a progressive disorder remains as yet unresolved. We previously demonstrated in a rat model of acquired focal cortical dysplasia, the methylazoxymethanol/pilocarpine - MAM/pilocarpine - rats, that the occurrence of status epilepticus (SE) and subsequent seizures fostered a pathologic process capable of modifying the morphology of cortical pyramidal neurons and NMDA receptor expression/localization. We have here extended our analysis by evaluating neocortical and hippocampal changes in MAM/pilocarpine rats at different epilepsy stages, from few days after onset up to six months of chronic epilepsy. Our findings indicate that the process triggered by SE and subsequent seizures in the malformed brain i) is steadily progressive, deeply altering neocortical and hippocampal morphology, with atrophy of neocortex and CA regions and progressive increase of granule cell layer dispersion; ii) changes dramatically the fine morphology of neurons in neocortex and hippocampus, by increasing cell size and decreasing both dendrite arborization and spine density; iii) induces reorganization of glutamatergic and GABAergic networks in both neocortex and hippocampus, favoring excitatory vs inhibitory input; iv) activates NMDA regulatory subunits. Taken together, our data indicate that, at least in experimental models of brain malformations, severe seizure activity, i.e., SE plus recurrent seizures, may lead to a widespread, steadily progressive architectural, neuronal and synaptic reorganization in the brain. They also suggest the mechanistic relevance of glutamate/NMDA hyper-activation in the seizure-related brain pathologic plasticity.

Long-duration epilepsy affects cell morphology and glutamatergic synapses in type IIB focal cortical dysplasia.

To investigate hypothesized effects of severe epilepsy on malformed cortex, we analyzed surgical samples from eight patients with type IIB focal cortical dysplasia (FCD) in comparison with samples from nine non-dysplastic controls. We investigated, using stereological quantification methods, where appropriate, dysplastic neurons, neuronal density, balloon cells, glia, glutamatergic synaptic input, and the expression of N-methyl-D-aspartate (NMDA) receptor subunits and associated membrane-associated guanylate kinase (MAGUK). In all FCD patients, the dysplastic areas giving rise to epileptic discharges were characterized by larger dysmorphic neurons, reduced neuronal density, and increased glutamatergic inputs, compared to adjacent areas with normal cytology. The duration of epilepsy was found to correlate directly (a) with dysmorphic neuron size, (b) reduced neuronal cell density, and (c) extent of reactive gliosis in epileptogenic/dysplastic areas. Consistent with increased glutamatergic input, western blot revealed that NMDA regulatory subunits and related MAGUK proteins were up-regulated in epileptogenic/dysplastic areas of all FCD patients examined. Taken together, these results support the hypothesis that epilepsy itself alters morphology-and probably also function-in the malformed epileptic brain. They also suggest that glutamate/NMDA/MAGUK dysregulation might be the intracellular trigger that modifies brain morphology and induces cell death.