RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0158295.].
RESUMO
Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit.
Assuntos
Cerebelo , Células de Purkinje , Animais , Camundongos , Encéfalo , Cognição , Camundongos Knockout , Fosfotirosina , Proteínas Tirosina Fosfatases , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genéticaRESUMO
Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells.
Assuntos
Caderinas , Sinapses , Animais , Proteínas de Ligação ao Cálcio , Cerebelo/fisiologia , Proteínas de Membrana , Camundongos , Células de Purkinje/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Diverse signaling complexes are precisely assembled at the presynaptic active zone for dynamic modulation of synaptic transmission and synaptic plasticity. Presynaptic GABAB-receptors nucleate critical signaling complexes regulating neurotransmitter release at most synapses. However, the molecular mechanisms underlying assembly of GABAB-receptor signaling complexes remain unclear. Here we show that neurexins are required for the localization and function of presynaptic GABAB-receptor signaling complexes. At four model synapses, excitatory calyx of Held synapses in the brainstem, excitatory and inhibitory synapses on hippocampal CA1-region pyramidal neurons, and inhibitory basket cell synapses in the cerebellum, deletion of neurexins rendered neurotransmitter release significantly less sensitive to GABAB-receptor activation. Moreover, deletion of neurexins caused a loss of GABAB-receptors from the presynaptic active zone of the calyx synapse. These findings extend the role of neurexins at the presynaptic active zone to enabling GABAB-receptor signaling, supporting the notion that neurexins function as central organizers of active zone signaling complexes.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptores de GABA-B/metabolismo , Sinapses/metabolismo , Animais , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cerebelo/citologia , Cerebelo/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa/genética , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Técnicas Estereotáxicas , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Neurexins are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. Here we identify FAM19A/TAFA proteins, "orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1γ, via an unusual mechanism. Specifically, we show that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type-specific regulators of neurexin modifications.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Quimiocinas/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Hipocampo/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismoRESUMO
To achieve ultrafast neurotransmission, neurons assemble synapses with highly organized presynaptic and postsynaptic nanomachines that are aligned by synaptic adhesion molecules. How functional assembly of presynaptic active zones is controlled via trans-synaptic interactions remains unknown. Here, we conditionally deleted all three neurexin adhesion molecules from presynaptic neurons of the calyx of Held in the mouse auditory system, a model synapse that allows precise biophysical analyses of synaptic properties. The pan-neurexin deletion had no effect on synapse development or the basic release machinery, but dramatically impaired fast neurotransmitter release. The overall properties of presynaptic calcium ion channels appeared normal, as reflected by the similar characteristics of calcium currents recorded at the nerve terminals. However, the pan-neurexin deletion significantly impaired the tight coupling of calcium influx to exocytosis, thereby suppressing neurotransmitter release. Furthermore, the pan-neurexin deletion reduced the function of calcium-activated BK potassium channels, whose activation depends on their tight association with presynaptic calcium channels. Together, these results suggest that neurexins perform a major function at the calyx synapse in coupling presynaptic calcium channels to release sites.
Assuntos
Canais de Cálcio/metabolismo , Proteínas do Tecido Nervoso/genética , Sinapses/metabolismo , Animais , Exocitose , Deleção de Genes , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transmissão SinápticaRESUMO
LAR-type receptor phosphotyrosine-phosphatases (LAR-RPTPs) are presynaptic adhesion molecules that interact trans-synaptically with multitudinous postsynaptic adhesion molecules, including SliTrks, SALMs, and TrkC. Via these interactions, LAR-RPTPs are thought to function as synaptogenic wiring molecules that promote neural circuit formation by mediating the establishment of synapses. To test the synaptogenic functions of LAR-RPTPs, we conditionally deleted the genes encoding all three LAR-RPTPs, singly or in combination, in mice before synapse formation. Strikingly, deletion of LAR-RPTPs had no effect on synaptic connectivity in cultured neurons or in vivo, but impaired NMDA-receptor-mediated responses. Deletion of LAR-RPTPs decreased NMDA-receptor-mediated responses by a trans-synaptic mechanism. In cultured neurons, deletion of all LAR-RPTPs led to a reduction in synaptic NMDA-receptor EPSCs, without changing the subunit composition or the protein levels of NMDA-receptors. In vivo, deletion of all LAR-RPTPs in the hippocampus at birth also did not alter synaptic connectivity as measured via AMPA-receptor-mediated synaptic responses at Schaffer-collateral synapses monitored in juvenile mice, but again decreased NMDA-receptor mediated synaptic transmission. Thus, LAR-RPTPs are not essential for synapse formation, but control synapse properties by regulating postsynaptic NMDA-receptors via a trans-synaptic mechanism that likely involves binding to one or multiple postsynaptic ligands.
Assuntos
Neurônios/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , TranscriptomaRESUMO
The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca2+-triggered synaptic vesicle exocytosis. BK-channels are Ca2+-activated large-conductance K+-channels that require close proximity to Ca2+-channels for activation and control Ca2+-triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK-channels are recruited to presynaptic Ca2+-channels, however, is unknown. Here, we show that RBPs (for RIM-binding proteins), which are evolutionarily conserved active zone proteins containing SH3- and FN3-domains, directly bind to BK-channels. We find that RBPs interact with RIMs and Ca2+-channels via their SH3-domains, but to BK-channels via their FN3-domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK-currents and depleted BK-channels from active zones. Our data suggest that RBPs recruit BK-channels into a RIM-based macromolecular active zone complex that includes Ca2+-channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio-temporal coupling of Ca2+-influx to Ca2+-triggered neurotransmitter release in a presynaptic terminal.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Canais de Cálcio/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Camundongos , Vesículas Sinápticas/genética , Domínios de Homologia de srcRESUMO
Recently a range of ocular manifestations such as retinal and lens amyloid-beta accumulation and retinal nerve fiber layer loss have been proposed as potential biomarkers in Alzheimer disease (AD). The TgCRND8 mouse model of AD exhibits age-dependent amyloid ß (Aß) oligomers accumulation and cognitive defects, amyloid plaques and hyperphosphorylated Tau deposition and inflammation. We proved the correlation between ocular pathologies and AD, observing increased levels of p-APP and p-Tau, accumulation of Aß oligomers in the retina, eye, and optic nerve. The accumulation of amyloid markers was significantly stronger in the retinal ganglion cell (RGC) layer, suggesting that RGC might be more susceptible to degeneration. We detected a thinning of the RGC layer as well as RGC death in the retina of TgCRND8 mice, by using a combination of Optical Coherence Tomography (OCT), immunofluorescence, immunohistochemistry and Western blotting techniques. We proved for the first time the key role of C-Jun N-terminal Kinase (JNK) in the ocular degeneration. In support of this, the administration of the JNK inhibitor, D-JNKI1, was able to counteract the Aß and p-Tau accumulation in the retina of TgCRND8 mice, and consequently reduce RGCs loss. These results confirm that degenerative changes in the retina/eye of AD mouse model mirrors the events observed in the brain parenchyma. Ocular changes can be detected by non-invasive imaging techniques, such as OCT, to study and test different therapeutic strategies against degenerative events associated to AD.
RESUMO
The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity.
Assuntos
Córtex Cerebral/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Terminações Pré-Sinápticas/enzimologia , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas SNARE/metabolismo , Animais , Córtex Cerebral/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Glicina/farmacologia , Masculino , Camundongos , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , N-Metilaspartato/farmacologia , Sinaptossomos/metabolismoRESUMO
Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges.
Assuntos
Retículo Endoplasmático/metabolismo , Fertilidade/genética , Fenótipo , Sinaptotagminas/deficiência , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Sobrevivência Celular/genética , Técnicas de Inativação de Genes , Ordem dos Genes , Marcação de Genes , Loci Gênicos , Genótipo , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Estresse Fisiológico , Sinaptotagminas/genéticaRESUMO
Alzheimer disease (AD) is the most prevalent form of dementia. Loss of hippocampal synapses is the first neurodegenerative event in AD. Synaptic loss has been associated with the accumulation in the brain parenchyma of soluble oligomeric forms of amyloid ß peptide (Aß1-42wt). Clinical observations have shown that a mutation in the APP protein (A673V) causes an early onset AD-type dementia in homozygous carriers while heterozygous carriers are unaffected. This mutation leads to the formation of mutated Aß peptides (Aß1-42A2V) in homozygous patients, while in heterozygous subjects both Aß1-42wt and Aß1-42A2V are present. To better understand the impact of the A673V mutation in AD, we analyzed the synaptotoxic effect of oligomers formed by aggregation of different Aß peptides (Aß1-42wt or Aß1-42A2V) and the combination of the two Aß1-42MIX (Aß1-42wt and Aß1-42A2V) in an in vitro model of synaptic injury. We showed that Aß1-42A2V oligomers are more toxic than Aß1-42wt oligomers in hippocampal neurons, confirming the results previously obtained in cell lines. Furthermore, we reported that oligomers obtained by the combination of both wild type and mutated peptides (Aß1-42MIX) did not exert synaptic toxicity. We concluded that the combination of Aß1-42wt and Aß1-42A2V peptides hinders the toxicity of Aß1-42A2V and counteracts the manifestation of synaptopathy in vitro. Finally we took advantage of this finding to generate a cell-permeable peptide for clinical application, by fusing the first six residues of the Aß1-42A2V to the TAT cargo sequence (Aß1-6A2VTAT(D)). Noteworthy, the treatment with Aß1-6A2VTAT(D) confers neuroprotection against both in vitro and in vivo synaptopathy models. Therefore Aß1-6A2VTAT(D) may represent an innovative therapeutic tool to prevent synaptic degeneration in AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/toxicidade , Hipocampo/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/toxicidade , Sinapses/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Animais , Permeabilidade da Membrana Celular , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/ultraestrutura , Sinapses/efeitos dos fármacosRESUMO
To characterize the mechanisms underlying region- and age-dependent hippocampal synaptic dysfunction in Alzheimer's disease, we used transgenic CRND8 mice, expressing the Swedish-Indiana APP mutation. In 2-month-old mice, no ß-amyloid plaques deposition, but the presence of soluble oligomers, were found in CA1 area but not in dentate gyrus (DG). At this age, long-term potentiation (LTP) was reduced selectively in CA1. In 6-month-old mice, the presence of soluble oligomers was accompanied by accumulation of ß-amyloid plaques and decreased LTP in CA1 and DG regions. In both regions, the loss of LTP was linked to reduced N-methyl-D-aspartate (NMDA) to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current ratio. The acetylcholine-esterase inhibitor, neostigmine rescued LTP in CA1 area at early stage of the disease but not after plaques deposition. Conversely, the NMDA receptor antagonist memantine restored LTP selectively in DG at later stages of the disease. Both these effects were associated with a normalization of the NMDA to AMPA ratio. The association between the recovery of LTP and the normalization of the NMDA to AMPA ratio provides information on new possible therapeutic strategies in Alzheimer's disease.
Assuntos
Envelhecimento/genética , Envelhecimento/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Potenciação de Longa Duração/genética , N-Metilaspartato/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Animais , Inibidores da Colinesterase/farmacologia , Modelos Animais de Doenças , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos Transgênicos , Terapia de Alvo Molecular , Mutação , Neostigmina/farmacologia , Placa Amiloide/metabolismoRESUMO
INTRODUCTION: Cell permeable peptides (CPPs) represent a novel tool for the delivery of bioactive molecules into scarcely accessible organs, such as the brain. CPPs have been successfully used in pre-clinical studies for a variety of diseases, ranging from cancer to neurological disorders. However, the mechanisms by which CPPs cross biological membranes, as well as their pharmacokinetic properties, have been poorly explored due to the lack of specific and sensitive analytical methods. METHODS: In this paper we describe a protocol to quantitatively determine the amount of CPPs in in vitro and in vivo experimental models. To this end we selected the peptide D-JNKI1 that was shown to prevent neurodegeneration in both acute and chronic degenerative disorders. This method allows an accurate quantitative analysis of D-JNKI1 in both neuronal lysates and tissue homogenates using mass spectrometry and stable isotope dilution approach. RESULTS: We found that D-JNKI1 crosses cellular membranes with fast kinetics, through an active and passive mechanism. After acute intraperitoneal (ip) administration of D-JNKI1 in mice, the peptide was found in the main organs with particular regard to the liver and kidney. Interestingly, D-JNKI1 crosses the blood brain barrier (BBB) and reaches the brain, where it remains for one week. DISCUSSION: The challenge lies in developing the clinical application of therapeutic cell permeable peptides. Discerning pharmacokinetic properties is a high priority to produce a powerful therapeutic strategy. Overall, our data shed light on the pharmacokinetic properties of D-JNKI1 and supports its powerful neuroprotective effect.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Animais , Animais Recém-Nascidos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Peptídeos/antagonistas & inibidoresAssuntos
Peptídeos beta-Amiloides/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Doença de Alzheimer , Peptídeos beta-Amiloides/farmacologia , Animais , Caspase 3/metabolismo , Caspase 3/fisiologia , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Modelos Biológicos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de SinaisRESUMO
The concept of "prion-like" has been proposed to explain the pathogenic mechanism of the principal neurodegenerative disorders associated with protein misfolding, including Alzheimer disease (AD). Other evidence relates prion protein with AD: the cellular prion protein (PrP(C)) binds ß amyloid oligomers, allegedly responsible for the neurodegeneration in AD, mediating their toxic effects. We and others have confirmed the high-affinity binding between ß amyloid oligomers and PrP(C), but we were not able to assess the functional consequences of this interaction using behavioral investigations and in vitro tests. This discrepancy rather than being resolved with the classic explanations, differencies in methodological aspects, has been reinforced by new data from different sources. Here we present data obtained with PrP antibody that not interfere with the neurotoxic activity of ß amyloid oligomers. Since the potential role of the PrP(C) in the neuronal dysfunction induced by ß amyloid oligomers is an important issue, find reasonable explanation of the inconsistent results is needed. Even more important however is the relevance of this interaction in the context of the disease, so as to develop valid therapeutic strategies.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Príons/metabolismo , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/metabolismo , Humanos , Príons/análiseRESUMO
A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-ß (Aß) peptide in the brain. Considerable evidence suggests that soluble Aß oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aß oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aß polymerization into amyloid fibrils. The ability of N1 to bind Aß oligomers is influenced by positively charged residues in two sites (positions 23-31 and 95-105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aß oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aß-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aß oligomer toxicity and represent an entirely new class of therapeutic agents for AD.
Assuntos
Peptídeos beta-Amiloides/química , Príons/química , Doença de Alzheimer/metabolismo , Proteínas Amiloidogênicas/química , Animais , Benzotiazóis , Caenorhabditis elegans/metabolismo , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Sinapses/metabolismo , Tiazóis/químicaRESUMO
Limiting the development of secondary damage represents one of the major goals of neuroprotective therapies after spinal cord injury. Here, we demonstrate that specific JNK inhibition via a single intraperitoneal injection of the cell permeable peptide D-JNKI1 6h after lesion improves locomotor recovery assessed by both the footprint and the BMS tests up to 4 months post-injury in mice. JNK inhibition prevents c-jun phosphorylation and caspase-3 cleavage, has neuroprotective effects and results in an increased sparing of white matter at the lesion site. Lastly, D-JNKI1 treated animals show a lower increase of erythrocyte extravasation and blood brain barrier permeability, thus indicating protection of the vascular system. In total, these results clearly point out JNK inhibition as a promising neuroprotective strategy for preventing the evolution of secondary damage after spinal cord injury.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Locomoção/efeitos dos fármacos , Fármacos Neuroprotetores , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Western Blotting , Caspase 3/metabolismo , Membro Posterior/fisiologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Imuno-Histoquímica , Injeções Intraperitoneais , Masculino , Camundongos , Fibras Nervosas/fisiologia , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-jun/metabolismo , Serotonina/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
BACKGROUND: Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H(2)O(2) induced-oxidative stress. Our data show that H(2)O(2) modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK's action with the D-JNKI1 peptide rescued cells from death. Following H(2)O(2), SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H(2)O(2) induced death. Importantly, in our model, direct interaction between these proteins can occur. CONCLUSIONS/SIGNIFICANCE: Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.
Assuntos
Peróxido de Hidrogênio/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação , Estresse Oxidativo , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de TempoRESUMO
Alzheimer disease (AD) is characterized by cognitive impairment that starts with memory loss to end in dementia. Loss of synapses and synaptic dysfunction are closely associated with cognitive impairment in AD patients. Biochemical and pathological evidence suggests that soluble Aß oligomers correlate with cognitive impairment. Here, we used the TgCRND8 AD mouse model to investigate the role of JNK in long term memory deficits. TgCRND8 mice were chronically treated with the cell-penetrating c-Jun N-terminal kinase inhibitor peptide (D-JNKI1). D-JNKI1, preventing JNK action, completely rescued memory impairments (behavioral studies) as well as the long term potentiation deficits of TgCRND8 mice. Moreover, D-JNKI1 inhibited APP phosphorylation in Thr-668 and reduced the amyloidogenic cleavage of APP and Aß oligomers in brain parenchyma of treated mice. In conclusion, by regulating key pathogenic mechanisms of AD, JNK might hold promise as innovative therapeutic target.