CNS autoimmune response in the MAM/pilocarpine rat model of epileptogenic cortical malformation.

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.

Peripheral blood mononuclear cell activation sustains seizure activity.

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)-1ß, 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.

pCREB expression in human tissues from epilepsy surgery.

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.

Epileptiform activity contralateral to unilateral hippocampal sclerosis does not cause the expression of brain damage markers.

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.

Kainic acid-induced albumin leak across the blood-brain barrier facilitates epileptiform hyperexcitability in limbic regions.

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 µm 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)-1ß 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.

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.

BET inhibitors (BETi) influence oxidative phosphorylation metabolism by affecting mitochondrial dynamics leading to alterations in apoptotic pathways in triple-negative breast cancer (TNBC) cells.

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.

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.

Intrinsic epileptogenicity of dysplastic cortex: converging data from experimental models and human patients.

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.

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron.

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.

Status epilepticus-induced pathologic plasticity in a rat model of focal cortical dysplasia.

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.

Unveiling Leukocyte Extracellular Traps in Inflammatory Responses of the Central Nervous System.

Over the past nearly two decades, increasing evidence has uncovered how immune cells can actively extrude genetic material to entrap invading pathogens or convey sterile inflammatory signals that contribute to shaping immune responses. Originally identified in neutrophils, the release of decondensed chromatin fibers decorated with antimicrobial proteins, called extracellular traps (ETs), has been recognized as a specific form of programmed inflammatory cell death, which is now known to occur in several other leukocytes. Subsequent reports have shown that self-DNA can be extruded from immune cells even in the absence of cell death phenomena. More recent data suggest that ETs formation could exacerbate neuroinflammation in several disorders of the central nervous system (CNS). This review article provides an overview of the varied types, sources, and potential functions of extracellular DNA released by immune cells. Key evidence suggesting the involvement of ETs in neurodegenerative, traumatic, autoimmune, and oncological disorders of the CNS will be discussed, outlining ongoing challenges and drawing potentially novel lines of investigation.

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.

Artificial neural networks allow the use of simultaneous measurements of Alzheimer disease markers for early detection of the disease.

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, sine-net, 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.

Pre-clinical diagnosis of Alzheimer disease combining platelet amyloid precursor protein ratio and rCBF spect analysis.

Platelet Amyloid Precursor Protein ratio of different abnormal forms and 99mTc-ECD SPECT perfusion analysis were evaluated in Mild Cognitive Impairment (MCI) subjects who progressed to Alzheimer Disease (AD) and in stable MCI. We report that their combined evaluation increases the discriminative power of the analysis in identifying presymptomatic AD. The positive predictive value of these combined markers in identifying progressive MCI was 0.87, and the negative predictive value was 0.90. This observation suggests that the interplay of different markers should be considered for enhancing diagnostic accuracy of pre-clinical AD.

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.

High cholesterol affects platelet APP processing in controls and in AD patients.

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 Abeta 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.

Abnormalities in the pattern of platelet amyloid precursor protein forms in patients with mild cognitive impairment and Alzheimer disease.

CONTEXT: Patients affected by sporadic Alzheimer disease (AD) show a significant alteration of amyloid precursor protein (APP) forms in platelets when compared with patients with dementia but without AD and age-matched controls. OBJECTIVE: To evaluate the ratio of platelet APP forms (APPr) in early-stage AD and mild cognitive impairment (MCI) and its potential as a biomarker for the early identification of AD. SETTING: Community population-based sample of patients admitted to 4 AD centers for investigation of cognitive disturbances. DESIGN AND METHODS: Thirty-five patients with mild AD (mAD), 21 patients with very mild AD (vmAD), 30 subjects with MCI, and 25 age-matched controls were included. The APPr was evaluated by Western blot analysis in platelet homogenate. RESULTS: Compared with controls (mean +/- SD, 0.93 +/- 0.3), the mean APPr was decreased in patients with mAD (0.44 +/- 0.24; P<.001) and patients with vmAD (0.49 +/- 0.3; P<.001). Regarding the MCI group, a significant decrease in APPr was found compared with controls (0.62 +/- 0.33; P<.001). Fixing a cutoff score of 0.6, sensitivity was 88.6% (31/35) for patients with mAD and 85.7% (18/21) for patients with vmAD, whereas specificity was 88% (22/25) for controls. Among patients with MCI, 18 (60%) of 30 individuals displayed APPr values below the cutoff. CONCLUSIONS: Alteration of platelet APP forms is an early event in AD, and the measurement of APPr may be useful for the identification of preclinical AD in patients with MCI.