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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. • SE induces PSD95-nNOS interaction, nitrosative stress, c-fos expression, spectrin degradation. • Tat-N-dimer peptide provides neuroprotective effect against status epilepticus. • Tat-N-dimer rescues the MP rats from SE-induced mortality. • Tat-N-dimer affects c-fos expression and neuronal death. • Tat-N-dimer affects SE-induced cytoskeletal degradation and hypergliosis [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Focal cortical dysplasia (FCD) is a brain malformation associated with particularly severe drug-resistant epilepsy that often requires surgery for seizure control. The molecular basis for such enhanced propensity to seizure generation in FCD is not as yet elucidated. To investigate cellular and molecular bases of epileptogenic mechanisms and possible effect of severe epilepsy on the malformed cortex we have here performed a parallel analysis of a rat model of acquired cortical dysplasia previously established in our laboratory, i.e., the methylazoxymethanol/pilocarpine (MAM-PILO) rats, and surgical samples from patients with type IIB FCD. Data from the MAM- PILO rat model and human FCD samples reveal in both conditions: (1) that status epilepticus ( SE) and/or seizures can further modify the cellular and molecular settings of the malformed cortex; (2) excitation/inhibition imbalance, and dysregulation of the N-methyl- d-aspartate/ membrane-associated guanylate kinase ( NMDA/ MAGUK) expression; (3) activation of cell death in neurons and glia. The data therefore highlight the mechanistic relevance of glutamate/ NMDA hyperactivation in FCD epileptogenesis and suggest that epilepsy is a pathologic process capable of affecting structure and function of both neurons and glia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have generated an experimental ‘double-hit’ model of chronic epilepsy to recapitulate the co-existence of abnormal cortical structure and frequently recurrent seizures as observed in human focal cortical dysplasia. We induced cortical malformations by exposing rats prenatally to methylazoxymethanol acetate and triggered status epilepticus and recurrent seizures in adult methylazoxymethanol acetate rats with pilocarpine. We studied the course of epilepsy and the long-term morphologic and molecular changes induced by the occurrence of status epilepticus and subsequent chronic epilepsy in the malformed methylazoxymethanol acetate exposed brain. Behavioural and electroencephalographic analyses showed that methylazoxymethanol acetate pilocarpine rats develop more severe epilepsy than naïve rats. Morphologic and molecular analyses demonstrated that status epilepticus and subsequent seizures, but not pilocarpine treatment per se, was capable of affecting both cortical architectural and N-methyl-d-aspartate receptor abnormalities induced by methylazoxymethanol acetate. In particular, cortical thickness was further decreased and N-methyl-d-aspartate regulatory subunits were recruited at the postsynaptic membrane. In addition, methylazoxymethanol acetate pilocarpine rats showed abnormally large cortical pyramidal neurons with neurofilament over-expression. These neurons bear similarities to the hypertrophic/dysmorphic pyramidal neurons observed in acquired human focal cortical dysplasia. These data show that status epilepticus sets in motion a pathological process capable of significantly changing the cellular and molecular features of pre-existing experimental cortical malformations. They suggest that seizure recurrence in human focal cortical dysplasia might be an additional factor in establishing a pathological circuitry that favours chronic neuronal hyperexcitability. [ABSTRACT FROM PUBLISHER]

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterised by a progressive cognitive and memory decline. From a neuropathological point of view, Alzheimer disease is defined by the presence of characteristic lesions, i.e. mature senile plaques, neurofibrillary tangles and amyloid angiopathy. In particular, accumulation of the amyloid β-peptide in the brain parenchyma and vasculature is an invariant event in the pathogenesis of both sporadic and familial Alzheimer cases. Amyloid β-peptide originates from a larger precursor, the Amyloid Precursor Protein (APP) ubiquitously expressed. Among the different peripheral cells expressing APP forms, platelets are particularly interesting since they show concentrations of its isoforms equivalent to those found in brain. Moreover, a number of laboratories independently described alterations in APP metabolism/concentration in platelets of Alzheimer patients when compared to control subjects matched for demographic characteristics. These observations defined the frame of our work aimed to investigate if a correlation between levels of platelet APP forms and Alzheimer disease could be detected. We have reported that patients affected by Alzheimer disease show a differential level of platelet APP forms. This observation has several implications: APP processing abnormalities, believed to be a very early change in Alzheimer disease in neuronal compartment, does occur in extraneuronal tissues, such as platelets, thus suggesting that Alzheimer disease is a systemic disorder; further, our data strongly indicate that a differential level of platelet APP forms can be considered a potential peripheral marker of Alzheimer disease allowing for discrimination between Alzheimer and other types of dementia with good sensitivity and specificity. [Copyright &y& Elsevier]

Clinical trials report that Ginkgo biloba extracts (e.g., EGb761) reduce cognitive symptoms in age-associated memory impairment and dementia, including Alzheimer disease (AD). However, the mechanisms behind their neuroprotective ability remain to be fully established.In this study, the effect of EGb761 on the amyloid precursor protein (APP) metabolism has been investigated by both in vitro and in vivo models. To this aim, alpha-secretase, the enzyme regulating the non-amyloidogenic processing of APP and the release of alphaAPPs, the alpha-secretase metabolite, were studied in superfusates of hippocampal slices after EGb761 incubation, and in hippocampi and cortices of EGb761-treated rats. PKC translocation state was evaluated as well.EGb761 increases alphaAPPs release through a PKC-independent manner. This effect is not accompanied by a modification of either APP forms or alpha-secretase expression. Moreover, EGb761 influence on alphaAPPs release was strictly dependent on treatment dosage.Our findings suggest that the benefit of EGb761 reported by previous clinical studies is underscored by a specific biological mechanism of this compound on APP metabolism, directly affecting the release of the non-amyloidogenic metabolite. Additional research will be needed to clearly define the effective clinical relevance, thus considering EGb761 as a possible supplementary treatment in dementing diseases. [Copyright &y& Elsevier]

Alzheimer disease (AD) is characterised by a decrease of platelet Amyloid Precursor Protein forms ratio (APPr), which parallels symptoms’ severity. Recent studies have suggested that cholesterol might play a role in the pathophysiology of AD by modulating Aβ production. Aim of this study was to evaluate the relationship between serum cholesterol levels and platelet APP processing in controls and AD.Sixty AD patients and 45 age-matched controls (CTRL) were investigated. Neuropsychological assessment, cholesterol dosage and APP forms’ evaluation were performed on each subject. CTRL showed lower serum cholesterol levels compared to AD (P<0.01) and higher mean APPr scores (P<0.0001). Hypercholesterolaemic AD patients showed lower APPr scores compared to normocholesterolaemic AD patients matched for disease severity (0.31±0.16 versus 0.45±0.28; P<0.05), since the early stage of the disease. In AD, cholesterol levels influence APPr independently of disease severity. These findings confirm the association between cholesterol and AD, and suggest that in vivo cholesterol affects APP processing by interfering with its maturation. [Copyright &y& Elsevier]

Three major amyloid precursor protein (APP) forms with apparent molecular weight ranging from 106 to 130 kDa are present in human platelets. Alzheimer disease (AD) is associated with a decreased APP forms ratio (APPr) between the three major forms. A total of 25 mild to moderate AD patients were investigated. Platelet APPr was studied before and after 30 days of acetylcholinesterase-inhibitor treatment (donepezil, 5 mg daily). Patients were grouped into non-∊4 carriers and ∊4 carriers according to apolipoprotein E (ApoE) genotype. At baseline, all patients showed low APPr levels and no significant difference was found between the two ApoE subgroups. After treatment, although a marked improvement in APPr was observed in most patients, non-∊4 carriers displayed a higher increase compared to ∊4 carriers (P<0.0001). The present study provides evidence that donepezil influences APP metabolism in platelets, and suggests that ApoE genotype might be an important modulating factor for drug responsiveness in AD. [Copyright &y& Elsevier]

Repressing BET proteins' function using bromodomain inhibitors (BETi) has been shown to elicit antitumor effects by regulating the transcription of genes downstream of BRD4. We previously showed that BETi promoted cell death of triple‐negative breast cancer (TNBC) cells. Here, we proved that BETi induce altered mitochondrial dynamics fitness in TNBC cells falling in cell death. We demonstrated that BETi treatment downregulated the expression of BCL‐2, and proteins involved in mitochondrial fission and increased fused mitochondria. Impaired mitochondrial fission affected oxidative phosphorylation (OXPHOS) inducing the expression of OXPHOS‐related genes, SDHa and ATP5a, and increased cell death. Consistently, the amount of mitochondrial DNA and mitochondrial membrane potential (∆Ψm) increased in BETi‐treated cells compared to control cells. Lastly, BETi in combination with Metformin reduced cell growth. Our results indicate that mitochondrial dynamics and OXPHOS metabolism support breast cancer proliferation and represent novel BETi downstream targets in TNBC cells. [ABSTRACT FROM AUTHOR]

Objective: The influx of immune cells and serum proteins from the periphery into the brain due to a dysfunctional blood‐brain barrier (BBB) has been proposed to contribute to the pathogenesis of seizures in various forms of epilepsy and encephalitis. We evaluated the pathophysiological impact of activated peripheral blood mononuclear cells (PBMCs) and serum albumin on neuronal excitability in an in vitro brain preparation. Methods: A condition of mild endothelial activation induced by arterial perfusion of lipopolysaccharide (LPS) was induced in the whole brain preparation of guinea pigs maintained in vitro by arterial perfusion. We analyzed the effects of co‐perfusion of human recombinant serum albumin with human PBMCs activated with concanavalin A on neuronal excitability, BBB permeability (measured by FITC‐albumin extravasation), and microglial activation. Results: Bioplex analysis in supernatants of concanavalin A–stimulated PBMCs revealed increased levels of several inflammatory mediators, in particular interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, interferon (INF)‐γ, IL‐6, IL‐10, IL‐17A, and MIP3α. LPS and human albumin arterially co‐perfused with either concanavalin A–activated PBMCs or the cytokine‐enriched supernatant of activated PBMCs (1) modulated calcium‐calmodulin–dependent protein kinase II at excitatory synapses, (2) enhanced BBB permeability, (3) induced microglial activation, and (4) promoted seizure‐like events. Separate perfusions of either nonactivated PBMCs or concanavalin A–activated PBMCs without LPS/human albumin (hALB) failed to induce inflammatory and excitability changes. Significance: Activated peripheral immune cells, such as PBMCs, and the extravasation of serum proteins in a condition of BBB impairment contribute to seizure generation. [ABSTRACT FROM AUTHOR]

The development of seizures in epilepsy syndromes associated with malformations of cortical development (MCDs) has traditionally been attributed to intrinsic cortical alterations resulting from abnormal network excitability. However, recent analyses at single-cell resolution of human brain samples from MCD patients have indicated the possible involvement of adaptive immunity in the pathogenesis of these disorders. By exploiting the MethylAzoxyMethanol (MAM)/pilocarpine (MP) rat model of drug-resistant epilepsy associated with MCD, we show here that the occurrence of status epilepticus and subsequent spontaneous recurrent seizures in the malformed, but not in the normal brain, are associated with the outbreak of a destructive autoimmune response with encephalitis-like features, involving components of both cell-mediated and humoral immune responses. The MP brain is characterized by blood-brain barrier dysfunction, marked and persisting CD8+ T cell invasion of the brain parenchyma, meningeal B cell accumulation, and complement-dependent cytotoxicity mediated by antineuronal antibodies. Furthermore, the therapeutic treatment of MP rats with the immunomodulatory drug fingolimod promotes both antiepileptogenic and neuroprotective effects. Collectively, these data show that the MP rat could serve as a translational model of epileptogenic cortical malformations associated with a central nervous system autoimmune response. This work indicates that a preexisting brain maldevelopment predisposes to a secondary autoimmune response, which acts as a precipitating factor for epilepsy and suggests immune intervention as a therapeutic option to be further explored in epileptic syndromes associated with MCDs. [ABSTRACT FROM AUTHOR]

Objective Systemic administration of kainic acid ( KA) is a widely used procedure utilized to develop a model of temporal lobe epilepsy ( TLE). Despite its ability to induce status epilepticus ( SE) in vivo, KA applied to in vitro preparations induces only interictal-like activity and/or isolated ictal discharges. The possibility that extravasation of the serum protein albumin from the vascular compartment enhances KA-induced brain excitability is investigated here. Methods Epileptiform activity was induced by arterial perfusion of 6 μ m KA in the in vitro isolated guinea pig brain preparation. Simultaneous field potential recordings were carried out bilaterally from limbic ( CA1, dentate gyrus [ DG], and entorhinal cortex) and extralimbic regions (piriform cortex and neocortex). Blood-brain barrier ( BBB) breakdown associated with KA-induced epileptiform activity was assessed by parenchymal leakage of intravascular fluorescein-isothiocyanate albumin. Seizure-induced brain inflammation was evaluated by western blot analysis of interleukin ( IL)-1β expression in brain tissue. Results KA infusion caused synchronized activity at 15-30 Hz in limbic (but not extralimbic) cortical areas, associated with a brief, single seizure-like event. A second bolus of KA, 60 min after the induction of the first ictal event, did not further enhance excitability. Perfusion of serum albumin between the two administrations of KA enhanced epileptiform discharges and allowed a recurrent ictal event during the second KA infusion. Significance Our data show that arterial KA administration selectively alters the synchronization of limbic networks. However, KA is not sufficient to generate recurrent seizures unless serum albumin is co-perfused during KA administration. These findings suggest a role of serum albumin in facilitating acute seizure generation. [ABSTRACT FROM AUTHOR]

Objective: Activity‐dependent changes have been reported in animal models and in human epileptic specimens and could potentially be used as tissue biomarkers to evaluate the propensity of a tissue to generate seizure activity. In this context, cAMP‐response element binding protein (CREB) activation was specifically reported in human epileptic foci and related mainly to interictal spike activity. To get further insights into CREB activation in human epilepsy, we analyzed pCREB expression on brain tissue samples from patients who underwent surgery for drug‐resistant focal epilepsy, correlating this expression with intracranial stereo–electroencephalography (SEEG) recording in a subgroup. Methods: Neocortical specimens from patients with neuropathological diagnosis of no lesion (cryptogenic), malformations of cortical development,mainly type II focal cortical dysplasia (FCD), and hippocampi with and without hippocampal sclerosis have been analyzed by immunohistochemistry. Peritumoral cortex from non‐epileptic patients and autoptic samples were used as controls, whereas rat brains were used to test possible loss of pCREB antigenicity due to fixation procedures and postmortem delay. Results: pCREB was consistently expressed in layer II neuronal nuclei in regions with normal cortical lamination both in epileptic and non‐epileptic surgical tissues. In patients with SEEG recordings, this anatomical pattern was unrelated to the presence of interictal spike activity. Conversely, in the core of type II FCD, as well as in other developmental malformations, pCREB was scattered without any laminar specificity. Furthermore, quantitative data did not reveal significant differences between epileptic and non‐epileptic tissues, except for an increased immunoreactivity in the core of type IIB FCD lesion related mainly to reactive glial and balloon cells. Significance: The present data argue against the reliability of pCREB immunohistochemistry as a marker of epileptic focus but underscores its layer‐related expression, suggesting a potential application in the study of malformations of cortical development, a wide range of diseases arising from perturbations of normal brain development. [ABSTRACT FROM AUTHOR]

Objective: Patients with epilepsy often ask if recurrent seizures harm their brain and aggravate their epileptic condition. This crucial question has not been specifically addressed by dedicated experiments. We analyze here if intense bilateral seizure activity induced by local injection of kainic acid (KA) in the right hippocampus produces brain damage in the left hippocampus. Methods: Adult guinea pigs were bilaterally implanted with hippocampal electrodes for continuous video—electroencephalography (EEG) monitoring. Unilateral injection of 1 μg KA in the dorsal CA1 area induced nonconvulsive status epilepticus (ncSE) characterized by bilateral hippocampal seizure discharges. This treatment resulted in selective unilateral sclerosis of the KA‐injected hippocampus. Three days after KA injection, the animals were killed, and the brains were submitted to ex vivo magnetic resonance imaging (MRI) and were processed for immunohistochemical analysis. Results: During ncSE, epileptiform activity was recorded for 27.6 ± 19.1 hours in both the KA‐injected and contralateral hippocampi. Enhanced T1‐weighted MR signal due to gadolinium deposition, mean diffusivity reduction, neuronal loss, gliosis, and blood–brain barrier permeability changes was observed exclusively in the KA‐injected hippocampus. Despite the presence of a clear unilateral hippocampal sclerosis at the site of KA injection, no structural alterations were detected by MR and immunostaining analysis performed in the hippocampus contralateral to KA injection 3 days and 2 months after ncSE induction. Fluoro‐Jade and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at the same time points confirmed the absence of degenerating cells in the hippocampi contralateral to KA injection. Significance: We demonstrate that intense epileptiform activity during ncSE does not cause obvious brain damage in the hippocampus contralateral to unilateral hippocampal KA injection. These findings argue against the hypothesis that epileptiform activity per se contributes to focal brain injury in previously undamaged cortical regions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 465-474. Abstract The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA. [ABSTRACT FROM AUTHOR]

Background: Previous studies have shown that in platelets of mild Alzheimer Disease (AD) patients there are alterations of specific APP forms, paralleled by alteration in expression level of both ADAM 10 and BACE when compared to control subjects. Due to the poor linear relation among each key-element of beta-amyloid cascade and the target diagnosis, the use of systems able to afford non linear tasks, like artificial neural networks (ANNs), should allow a better discriminating capacity in comparison with classical statistics. Objective: To evaluate the accuracy of ANNs in AD diagnosis. Methods: 37 mild-AD patients and 25 control subjects were enrolled, and APP, ADM10 and BACE measures were performed. Fifteen different models of feed-forward and complex-recurrent ANNs (provided by Semeion Research Centre), based on different learning laws (back propagation, sinenet, bi-modal) were compared with the linear discriminant analysis (LDA). Results: The best ANN model correctly identified mild AD patients in the 94% of cases and the control subjects in the 92%. The corresponding diagnostic performance obtained with LDA was 90% and 73%. Conclusion: This preliminary study suggests that the processing of biochemical tests related to beta-amyloid cascade with ANNs allows a very good discrimination of AD in early stages, higher than that obtainable with classical statistics methods. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimer's Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well-known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha-secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH-SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH-SY5Y cells released significantly more sAPPα into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPα levers. Pre-incubation with atropine decreased release of sAPPα significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis. [ABSTRACT FROM AUTHOR]

Accumulating evidence from epidemiological and clinical studies suggests that vascular risk factors may be involved in Alzheimer disease (AD). Although the precise contribution of vascular disturbances to the pathogenesis of AD is still unclear, various biochemical and neuropathological data strengthen the view that cerebrovascular deficiencies such as reduced blood supply to the brain and disrupted microvascular integrity in brain parenchyma play a direct or intermediate role in the chain of events ending with a dementia syndrome. The present review focuses on platelet abnormalities and hemostatic alterations in AD. In particular, data from our group, along with current literature, are discussed with regard to the evidence of platelets amyloid precursor protein (APP) processing disturbances in early AD as well as to the recent observations of increased serum levels of thrombomodulin and sE-selectin, which are sensitive markers of endothelial dysfunction. These findings strongly indicate that platelet dysfunction and microvasculature deficiencies occur rather early during the course of AD, thus suggesting a further link between AD-related processes and vascular disorders. [Copyright &y& Elsevier]