Neuroprotective and Antioxidant Properties of CholesteroNitrone ChN2 and QuinolylNitrone QN23 in an Experimental Model of Cerebral Ischemia: Involvement of Necrotic and Apoptotic Cell Death.

Ischemic stroke is the leading cause of disability and the second leading cause of death worldwide. However, current therapeutic strategies are scarce and of limited efficacy. The abundance of information available on the molecular pathophysiology of ischemic stroke has sparked considerable interest in developing new neuroprotective agents that can target different events of the ischemic cascade and may be used in combination with existing treatments. In this regard, nitrones represent a very promising alternative due to their renowned antioxidant and anti-inflammatory effects. In this study, we aimed to further investigate the neuroprotective effects of two nitrones, cholesteronitrone 2 (ChN2) and quinolylnitrone 23 (QN23), which have previously shown great potential for the treatment of stroke. Using an experimental in vitro model of cerebral ischemia, we compared their anti-necrotic, anti-apoptotic, and antioxidant properties with those of three reference compounds. Both ChN2 and QN23 demonstrated significant neuroprotective effects (EC50 = 0.66 ± 0.23 µM and EC50 = 2.13 ± 0.47 µM, respectively) comparable to those of homo-bis-nitrone 6 (HBN6) and N-acetylcysteine (NAC) and superior to those of α-phenyl-N-tert-butylnitrone (PBN). While primarily derived from the nitrones' anti-necrotic capacities, their anti-apoptotic effects at high concentrations and antioxidant powers-especially in the case of QN23-also contribute to their neuroprotective effects.

Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub.

Neuropsychiatric disorders (NPDs) are frequently co-morbid with epilepsy, but the biological basis of shared risk remains poorly understood. The 16p11.2 duplication is a copy number variant that confers risk for diverse NPDs including autism spectrum disorder, schizophrenia, intellectual disability and epilepsy. We used a mouse model of the 16p11.2 duplication (16p11.2dup/+) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics revealed alterations to synaptic networks and products of NPD risk genes. We identified an epilepsy-associated subnetwork that was dysregulated in 16p11.2dup/+ mice and altered in brain tissue from individuals with NPDs. Cortical circuits from 16p11.2dup/+ mice exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. Using gene co-expression and interactome analysis, we show that PRRT2 is a major hub in the epilepsy subnetwork. Remarkably, correcting Prrt2 copy number rescued aberrant circuit properties, seizure susceptibility and social deficits in 16p11.2dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic disorders, and reveal mechanisms relevant to the complex symptomatology of 16p11.2 duplication carriers.

A novel role for the late-onset Alzheimer's disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling.

Postsynaptic trafficking plays a key role in regulating synapse structure and function. While spiny excitatory synapses can be stable throughout adult life, their morphology and function is impaired in Alzheimer's disease (AD). However, little is known about how AD risk genes impact synaptic function. Here we used structured superresolution illumination microscopy (SIM) to study the late-onset Alzheimer's disease (LOAD) risk factor BIN1, and show that this protein is abundant in postsynaptic compartments, including spines. While postsynaptic Bin1 shows colocalization with clathrin, a major endocytic protein, it also colocalizes with the small GTPases Rab11 and Arf6, components of the exocytic pathway. Bin1 participates in protein complexes with Arf6 and GluA1, and manipulations of Bin1 lead to changes in spine morphology, AMPA receptor surface expression and trafficking, and AMPA receptor-mediated synaptic transmission. Our data provide new insights into the mesoscale architecture of postsynaptic trafficking compartments and their regulation by a major LOAD risk factor.

CNTNAP2 is targeted to endosomes by the polarity protein PAR3.

A decade of genetic studies has established contactin-associated protein-like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2's molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and partitioning-defective 3 (PAR3)-a polarity molecule isolated in a yeast two-hybrid screen with CNTNAP2's C-terminus. We provide evidence that the two proteins interact via PDZ domain-mediated binding, that CNTNAP2+ /PAR3+ complexes are largely associated with clathrin-coated endocytic vesicles in heterologous cells and that PAR3 causes an enlargement of CNTNAP2 puncta size. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that PAR3 limits the mobility of CNTNAP2. Finally, overexpression of PAR3 but not a PAR3 mutant lacking all PDZ domains (PAR3∆PDZall) can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that PAR3 regulates CNTNAP2 spatial localization.

The CNTNAP2-CASK complex modulates GluA1 subcellular distribution in interneurons.

GABAergic interneurons are emerging as prominent substrates in the pathophysiology of multiple neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, and epilepsy. Interneuron excitatory activity is influenced by 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptors (AMPARs), which in turn affects excitatory transmission in the central nervous system. Yet how dysregulation of interneuronal AMPARs distinctly contributes to the molecular underpinning of neurobiological disease is drastically underexplored. Contactin-associated protein-like 2 (CNTNAP2) is a neurexin-related adhesion molecule shown to mediate AMPAR subcellular distribution while calcium/calmodulin-dependent serine protein kinase (CASK) is a multi-functional scaffold involved with glutamate receptor trafficking. Mutations in both genes have overlapping disease associations, including autism spectrum disorders, intellectual disability, and epilepsy, thus suggesting converging perturbations of excitatory/inhibitory balance. Our lab has previously shown that CNTNAP2 stabilizes interneuron dendritic arbors through CASK and that CNTNAP2 regulates AMPAR subunit GluA1 trafficking in excitatory neurons. The interaction between these three proteins, however, has not been studied in interneurons. Using biochemical techniques, structured illumination microscopy (SIM) and shRNA technology, we first confirm that these three proteins interact in mouse brain, and then examined relationship between CNTNAP2, CASK and GluA1 in mature interneurons. Using SIM, we ascertain that a large fraction of endogenous CNTNAP2, CASK, and GluA1 molecules collectively colocalize together in a tripartite manner. Finally, individual knockdown of either CNTNAP2 or CASK similarly alter GluA1 levels and localization. These findings offer insight to molecular mechanisms underlying GluA1 regulation in interneurons.

CNTNAP2 stabilizes interneuron dendritic arbors through CASK.

Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated-emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2's C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching in particular cortical regions, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.

The APPswe/PS1A246E mutations in an astrocytic cell line leads to increased vulnerability to oxygen and glucose deprivation, Ca2+ dysregulation, and mitochondrial abnormalities.

Growing evidence suggests a close relationship between Alzheimer's Disease (AD) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APPSWE /PS1A246E (APP, amyloid precursor protein; PS1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD), an in vitro model of ischemia. Cell death was increased in the APPSWE /PS1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca2+ ]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca2+ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca2+ ] was measured by fluorescence microscopy. Changes in cytosolic Ca2+ concentration ([Ca2+ ]c ) were potentiated in the APPSWE /PS1A246E transgenic line. Mitochondrial function was also altered in the APPSWE /PS1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca2+ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders.

Folic Acid Protects Against Glutamate-Induced Excitotoxicity in Hippocampal Slices Through a Mechanism that Implicates Inhibition of GSK-3ß and iNOS.

Folic acid (folate) is a vitamin of the B-complex group crucial for neurological function. Considering that excitotoxicity and cell death induced by glutamate are involved in many disorders, the potential protective effect of folic acid on glutamate-induced cell damage in rat hippocampal slices and the possible intracellular signaling pathway involved in such effect were investigated. The treatment of hippocampal slices with folic acid (100 µM) significantly abrogated glutamate (1 mM)-induced reduction of cell viability measured by MTT reduction assay and inhibited glutamate-induced D-[3H]-aspartate release. To investigate the putative intracellular signaling pathways implicated in the protective effect of folic acid, we used a PI3K inhibitor, LY294002, which abolished the protective effects of folic acid against glutamate-induced cell damage and D-[3H] aspartate release. Moreover, hippocampal slices incubated with folic acid alone for 30 min presented increased phosphorylation of GSK-3ß at Ser9, indicating an inhibition of the activity of this enzyme. Furthermore, folic acid in the presence of glutamate insult in hippocampal slices maintained for an additional period of 6 h in fresh culture medium without glutamate and/or folic acid induced phosphorylation of GSK-3ß and ß-catenin expression. In addition, glutamate-treated hippocampal slices showed increased iNOS expression that was reversed by folic acid. In conclusion, the results of this study show that the protective effect of folic acid against glutamate-induced excitotoxicity may involve the modulation of PI3K/GSK-3ß/ß-catenin pathway and iNOS inhibition.

Reversal of dendritic phenotypes in 16p11.2 microduplication mouse model neurons by pharmacological targeting of a network hub.

The architecture of dendritic arbors contributes to neuronal connectivity in the brain. Conversely, abnormalities in dendrites have been reported in multiple mental disorders and are thought to contribute to pathogenesis. Rare copy number variations (CNVs) are genetic alterations that are associated with a wide range of mental disorders and are highly penetrant. The 16p11.2 microduplication is one of the CNVs most strongly associated with schizophrenia and autism, spanning multiple genes possibly involved in synaptic neurotransmission. However, disease-relevant cellular phenotypes of 16p11.2 microduplication and the driver gene(s) remain to be identified. We found increased dendritic arborization in isolated cortical pyramidal neurons from a mouse model of 16p11.2 duplication (dp/+). Network analysis identified MAPK3, which encodes ERK1 MAP kinase, as the most topologically important hub in protein-protein interaction networks within the 16p11.2 region and broader gene networks of schizophrenia-associated CNVs. Pharmacological targeting of ERK reversed dendritic alterations associated with dp/+ neurons, outlining a strategy for the analysis and reversal of cellular phenotypes in CNV-related psychiatric disorders.

Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons.

Central glutamatergic synapses and the molecular pathways that control them are emerging as common substrates in the pathogenesis of mental disorders. Genetic variation in the contactin associated protein-like 2 (CNTNAP2) gene, including copy number variations, exon deletions, truncations, single nucleotide variants, and polymorphisms have been associated with intellectual disability, epilepsy, schizophrenia, language disorders, and autism. CNTNAP2, encoded by Cntnap2, is required for dendritic spine development and its absence causes disease-related phenotypes in mice. However, the mechanisms whereby CNTNAP2 regulates glutamatergic synapses are not known, and cellular phenotypes have not been investigated in Cntnap2 knockout neurons. Here we show that CNTNAP2 is present in dendritic spines, as well as axons and soma. Structured illumination superresolution microscopy reveals closer proximity to excitatory, rather than inhibitory synaptic markers. CNTNAP2 does not promote the formation of synapses and cultured neurons from Cntnap2 knockout mice do not show early defects in axon and dendrite outgrowth, suggesting that CNTNAP2 is not required at this stage. However, mature neurons from knockout mice show reduced spine density and levels of GluA1 subunits of AMPA receptors in spines. Unexpectedly, knockout neurons show large cytoplasmic aggregates of GluA1. Here we characterize, for the first time to our knowledge, synaptic phenotypes in Cntnap2 knockout neurons and reveal a novel role for CNTNAP2 in GluA1 trafficking. Taken together, our findings provide insight into the biological roles of CNTNAP2 and into the pathogenesis of CNTNAP2-associated neuropsychiatric disorders.

Nrf2 participates in depressive disorders through an anti-inflammatory mechanism.

A causative relationship between inflammation and depression is gradually gaining consistency. Because Nrf2 participates in inflammation, we hypothesized that Nrf2 could play a role in depressive disorders. In this study, we have observed that Nrf2 deletion in mice results in: (i) a depressive-like behavior evaluated as an increase in the immobility time in the tail-suspension test and by a decrease in the grooming time in the splash test, (ii) reduced levels of dopamine and serotonin and increased levels of glutamate in the prefrontal cortex, (iii) altered levels of proteins associated to depression such as VEGF and synaptophysin and (iv) microgliosis. Furthermore, treatment of Nrf2 knockout mice with the anti-inflammatory drug rofecoxib reversed their depressive-like behavior, while induction of Nrf2 by sulforaphane, in an inflammatory model of depression elicited by LPS, afforded antidepressant-like effects. In conclusion, our results indicate that chronic inflammation due to a deletion of Nrf2 can lead to a depressive-like phenotype while induction of Nrf2 could become a new and interesting target to develop novel antidepressive drugs.

Involvement of PI3K, GSK-3ß and PPARγ in the antidepressant-like effect of folic acid in the forced swimming test in mice.

Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the pathophysiology of depression. Considering that alterations in the signaling pathways that regulate neuroplasticity and cellular survival are implicated in depressive disorders, the present study investigated the involvement of the phosphoinositide 3-kinase (PI3K), glycogen synthase kinase-3 (GSK-3ß), and peroxisome proliferator-activated receptor-γ (PPARγ) in the antidepressant-like effect of folic acid in the forced swimming test (FST). The intracerebroventricular (i.c.v.) pre-treatment of mice with LY294002 (10 nmol/site, a PI3K inhibitor) or GW-9662 (1 µg/site, a PPARγ antagonist) prevented the antidepressant-like effect of folic acid (50 mg/kg, p.o.) in the FST. In addition, the administration of subeffective doses of the selective GSK-3ß inhibitor, AR-A014418 (3 mg/kg, i.p.), a non-selective GSK-3ß inhibitor, lithium chloride (10 mg/kg, p.o) or a PPARγ agonist, rosiglitazone (1 µg/site, i.c.v.) in combination with a subeffective dose of folic acid (10 mg/kg, p.o.) significantly reduced the immobility time in the FST as compared with either drug alone, without altering the locomotor activity. These results indicate that the antidepressant-like effect of folic acid in the FST might be dependent on inhibition of GSK-3ß and activation of PPARγ, reinforcing the notion that these are important targets for antidepressant activity.

Neurotoxicity induced by okadaic acid in the human neuroblastoma SH-SY5Y line can be differentially prevented by α7 and ß2* nicotinic stimulation.

A good model of neuronal death that reproduces the characteristic tau (τ) hyperphosphorylation of Alzheimers disease is the use of okadaic acid (OA). The aim of this study was to determine the contribution of α7 and ß2* nicotinic acetylcholine receptor (nAChR) subtypes to neuroprotection against OA in the SH-SY5Y cell line by using the selective α7 and ß2* nAChR agonists PNU 282987 and 5-Iodo-A85380, respectively. The results of this study show that both α7 and ß2* nAChR can afford neuroprotection against OA-induced neurotoxicity. Protection mediated by α7 nAChRs was independent of Ca(2+) and involved the intracellular signaling pathway Janus Kinase-2/Phosphatidylinositol-3-kinase/Akt. When Ca(2+) entry was promoted through the α7 nAChR by using the α7-selective positive allosteric modulator PNU 120596, protection was lost. By contrast, protection mediated by ß2* nAChRs was Ca(2+) dependent and implicated the signaling pathways PI3K/Akt and extracellular regulated kinase 1/2. Both α7 and ß2* nAChR activation converged on downregulation of GSK-3ß and reduction of τ phosphorylation in cells undergoing cell death induced by OA. Therefore, targeting nAChR could offer a strategy for reducing neurodegeneration secondary to hyperphosphorylation of protein τ.

N-acylaminophenothiazines: neuroprotective agents displaying multifunctional activities for a potential treatment of Alzheimer's disease.

We have previously reported the multifunctional profile of N-(3-chloro-10H-phenothiazin-10-yl)-3-(dimethylamino)propanamide (1) as an effective neuroprotectant and selective butyrylcholinesterase inhibitor. In this paper, we have developed a series of N-acylaminophenothiazines obtained from our compound library or newly synthesised. At micro- and sub-micromolar concentrations, these compounds selectively inhibited butyrylcholinesterase (BuChE), protected neurons against damage caused by both exogenous and mitochondrial free radicals, showed low toxicity, and could penetrate into the CNS. In addition, N-(3-chloro-10H-phenothiazin-10-yl)-2-(pyrrolidin-1-yl)acetamide (11) modulated the cytosolic calcium concentration and protected human neuroblastoma cells against several toxics, such as calcium overload induced by an L-type Ca2+-channel agonist, tau-hyperphosphorylation induced by okadaic acid and Aß peptide.

Chondroitin sulfate reduces cell death of rat hippocampal slices subjected to oxygen and glucose deprivation by inhibiting p38, NFκB and iNOS.

The glycosaminoglycan chondroitin sulfate (CS) is a major constituent of the extracellular matrix of the central nervous system where it can constitute part of the perineuronal nets. Constituents of the perineuronal nets are gaining interest because they have modulatory actions on their neighbouring neurons. In this study we have investigated if CS could afford protection in an acute in vitro ischemia/reoxygenation model by using isolated hippocampal slices subjected to 60min oxygen and glucose deprivation (OGD) followed by 120min reoxygenation (OGD/Reox). In this toxicity model, CS afforded protection of rat hippocampal slices measured as a reduction of lactate dehydrogenase (LDH) release; maximum protection (70% reduction of LDH) was obtained at the concentration of 3mM. To evaluate the intracellular signaling pathways implicated in the protective effect of CS, we first analysed the participation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 by western blot. OGD/Reox induced the phosphorylation of p38 and dephosphorylation of ERK1/2; however, CS only inhibited p38 but had no effect on ERK1/2. Furthermore, OGD/Reox-induced translocation of p65 to the nucleus was prevented in CS treated hippocampal slices. Finally, CS inhibited iNOS induction caused by OGD/Reox and thereby nitric oxide (NO) production measured as a reduction in DAF-2 DA fluorescence. In conclusion, the protective effect of CS in hippocampal slices subjected to OGD/Reox can be related to a modulatory action of the local immune response by a mechanism that implies inhibition of p38, NFκB, iNOS and the production of NO.