RESUMO
A novel function for the neural cell adhesion molecule (NCAM) was identified in ephrinA/EphA-mediated repulsion as an important regulatory mechanism for development of GABAergic inhibitory synaptic connections in mouse prefrontal cortex. Deletion of NCAM, EphA3, or ephrinA2/3/5 in null mutant mice increased the numbers and size of perisomatic synapses between GABAergic basket interneurons and pyramidal cells in the developing cingulate cortex (layers II/III). A functional consequence of NCAM loss was increased amplitudes and faster kinetics of miniature inhibitory postsynaptic currents in NCAM null cingulate cortex. NCAM and EphA3 formed a molecular complex and colocalized with the inhibitory presynaptic marker vesicular GABA transporter (VGAT) in perisomatic puncta and neuropil in the cingulate cortex. EphrinA5 treatment promoted axon remodeling of enhanced green fluorescent protein-labeled basket interneurons in cortical slice cultures and induced growth cone collapse in wild-type but not NCAM null mutant neurons. NCAM modified with polysialic acid (PSA) was required to promote ephrinA5-induced axon remodeling of basket interneurons in cortical slices, likely by providing a permissive environment for ephrinA5/EphA3 signaling. These results reveal a new mechanism in which NCAM and ephrinAs/EphA3 coordinate to constrain GABAergic interneuronal arborization and perisomatic innervation, potentially contributing to excitatory/inhibitory balance in prefrontal cortical circuitry.
Assuntos
Efrinas/metabolismo , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Plasticidade Neuronal/fisiologia , Córtex Pré-Frontal/fisiologia , Sinapses/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Ácido gama-Aminobutírico/metabolismoRESUMO
The L1 adhesion molecule functions in axon growth and guidance, but a role in synaptic development of cortical inhibitory interneurons is largely unexplored. L1 mediates adhesion by engaging the actin cytoskeleton through binding the actin/spectrin adapter protein ankyrin. Loss of L1-ankyrin interaction impaired process elaboration/branching by GABAergic interneurons, including basket cells, and reduced the number of perisomatic synapses in the cingulate cortex as shown in L1 mutant mice (L1YH) with a mutation in the ankyrin-binding site, either alone or intercrossed with GAD67-enhanced green fluorescence protein reporter mice. Electron microscopy revealed that perisomatic inhibitory synapses but not excitatory synapses in the neuropil were specifically affected. In wild-type cingulate cortex, L1 colocalized with perisomatic synaptic markers, whereas L1 phosphorylation on Tyr(1229) decreased postnatally, correlating with increased ankyrin binding and synaptic development. These results suggest a novel role for L1 engagement with the actin cytoskeleton in development of inhibitory connectivity within the cingulate cortex.
Assuntos
Anquirinas/fisiologia , Interneurônios/metabolismo , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Córtex Pré-Frontal/fisiologia , Terminações Pré-Sinápticas/fisiologia , Ácido gama-Aminobutírico/fisiologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Técnicas de Introdução de Genes , Giro do Cíngulo/metabolismo , Giro do Cíngulo/fisiologia , Interneurônios/citologia , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/fisiologia , Molécula L1 de Adesão de Célula Nervosa/genética , Inibição Neural/genética , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Ligação Proteica/genética , Sinapses/genéticaRESUMO
Neprilysin (NEP) is a zinc metallopeptidase that efficiently degrades the amyloid beta (Abeta) peptides believed to be involved in the etiology of Alzheimer disease (AD). The focus of this study was to develop a new and tractable therapeutic approach for treating AD using NEP gene therapy. We have introduced adeno-associated virus (AAV) expressing the mouse NEP gene into the hindlimb muscle of 6-month-old human amyloid precursor protein (hAPP) (3X-Tg-AD) mice, an age which correlates with early stage AD. Overexpression of NEP in muscle decreased brain soluble Abeta peptide levels by approximately 60% and decreased amyloid deposits by approximately 50%, with no apparent adverse effects. Expression of NEP on muscle did not affect the levels of a number of other physiological peptides known to be in vitro substrates. These findings demonstrate that peripheral expression of NEP and likely other peptidases represents an alternative to direct administration into brain and illustrates the potential for using NEP expression in muscle for the prevention and treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Regulação da Expressão Gênica , Neprilisina/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Membro Posterior/metabolismo , Membro Posterior/patologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Neprilisina/genéticaRESUMO
A number of therapeutic strategies for treating Alzheimer's disease have focused on reducing amyloid burden in the brain. Among these approaches, the expression of amyloid beta peptide (Abeta)-degrading enzymes in the brain has been shown to be effective but to date not practical for treating patients. We report here a novel strategy for lowering amyloid burden in the brain by peripherally expressing the Abeta-degrading enzyme neprilysin on leukocytes in the 3xTg-AD mouse model of Alzheimer's disease. Through transplantation of lentivirus-transduced bone marrow cells, the Abeta-degrading protease neprilysin was expressed on the surface of leukocytes. This peripheral neprilysin reduced soluble brain Abeta peptide levels by approximately 30% and lowered the accumulation of amyloid beta peptides by 50-60% when transplantation was performed at both young and early adult age. In addition, peripheral neprilysin expression reduced amyloid-dependent performance deficits as measured by the Morris water maze. Unlike other methods designed to lower Abeta levels in blood, which cause a net increase in peptide, neprilysin expression results in the catabolism of Abeta to small, innocuous peptide fragments. These findings demonstrate that peripherally expressed neprilysin, and likely other Abeta-degrading enzymes, has the potential to be utilized as a therapeutic approach to prevent and treat Alzheimer's disease and suggest that this approach should be explored further.
Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Terapia Genética , Leucócitos/metabolismo , Neprilisina/genética , Neprilisina/metabolismo , Envelhecimento , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Pressão Sanguínea , Transplante de Medula Óssea , Transtornos Cognitivos/terapia , Modelos Animais de Doenças , Humanos , Lentivirus , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Transdução GenéticaRESUMO
It is generally believed that amyloid beta peptides (Abeta) are the key mediators of Alzheimer's disease. Therapeutic interventions have been directed toward impairing the synthesis or accelerating the clearance of Abeta. An equilibrium between blood and brain Abeta exists mediated by carriers that transport Abeta across the blood-brain barrier. Passive immunotherapy has been shown to be effective in mouse models of AD, where the plasma borne antibody binds plasma Abeta causing an efflux of Abeta from the brain. As an alternative to passive immunotherapy we have considered the use of Abeta-degrading peptidases to lower plasma Abeta levels. Here we compare the ability of three Abeta-degrading peptidases to degrade Abeta. Biotinylated peptidases were coupled to the surface of biotinylated erythrocytes via streptavidin. These erythrocyte-bound peptidases degrade Abeta peptide in plasma. Thus, peptidases bound to or expressed on the surface of erythroid cells represent an alternative to passive immunotherapy.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Eritrócitos/metabolismo , Peptídeo Hidrolases/metabolismo , Doença de Alzheimer/sangue , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/sangue , Animais , Biotinilação , Barreira Hematoencefálica , Humanos , Imunização Passiva , Técnicas In Vitro , Insulisina/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Neprilisina/metabolismo , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/uso terapêuticoRESUMO
Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.
Assuntos
Apoptose/genética , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Metaloendopeptidases/genética , Mitocôndrias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Masculino , Metaloendopeptidases/metabolismo , Metaloendopeptidases/farmacologia , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/metabolismo , RatosRESUMO
That neprilysin (NEP) is a major Abeta peptide-degrading enzyme in vivo is shown by higher Abeta peptide levels in the brain of an NEP knockout mouse. In addition, we show that infusion of an NEPinhibitor, but not inhibitors of other peptidases, into the brains of an APP transgenic mouse elevates Abeta levels. We have investigated the use of NEP as a potential therapeutic agent to prevent the accumulation of Abeta peptides in the brain. Lentivirus expressing NEP was initially used to demonstrate the ability of the enzyme to reduce Abeta levels in a model CHO cell line and to make primary hippocampal neurons resistant to Abeta-mediated neurotoxicity. Injection of NEPexpressing lentivirus, but not inactive NEP-expressing lentivirus, GFP-expressing lentivirus, or vehicle, into the hippocampus of 12-20-mo-old hAPP transgenic mice led to an approx 50% reduction in the number of amyloid plaques. These studies provide the impetus for further investigating of the use of NEP in a gene transfer therapy paradigm to prevent the accumulation of Abeta and prevent or delay the onset of Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Regulação para Baixo/genética , Neprilisina/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Resistência a Medicamentos/genética , Inibidores Enzimáticos/farmacologia , Feto , Terapia Genética/métodos , Vetores Genéticos , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neprilisina/antagonistas & inibidores , Neprilisina/genética , Placa Amiloide/genética , Placa Amiloide/metabolismoRESUMO
Neprilysin is a zinc metalloendopeptidase that regulates the activity of a number of physiological peptides through hydrolytic inactivation. Most recently, evidence has accumulated that neprilysin is involved in the clearance of amyloid beta peptides in the brain. Previous studies have shown that the neprilysin gene responds to progesterone, androgen, and glucocorticoids. We now show that estrogen regulates neprilysin activity in rat brain. Ovariectomy leads to a 30% decrease in neprilysin activity at 45 or 85 days, but not 21 days, post surgery. Estrogen replacement restores neprilysin levels back to control values. These changes in neprilysin activity suggest that in women estrogen is required to maintain basal levels of neprilysin.
Assuntos
Encéfalo/efeitos dos fármacos , Terapia de Reposição de Estrogênios/métodos , Estrogênios/farmacologia , Neprilisina/metabolismo , Análise de Variância , Animais , Encéfalo/anatomia & histologia , Encéfalo/enzimologia , Estrogênios/sangue , Feminino , Ovariectomia/métodos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations. RESULTS: An N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved Tau, product analysis with N and C terminal Tau antibodies coupled with mass spectrometry showed an endoproteolytic cleavage atypical for an aminopeptidase. Furthermore, the reaction was not blocked by the general aminopeptidase inhibitor bestatin or the specific PSA inhibitor puromycin. In order to test whether Tau hydrolysis might be caused by a protease contaminant the enzyme was expressed in E. coli as glutathione S-transferase and maltose binding protein fusion proteins or in Sf9 cells as a C-terminally His-tagged protein. After purification to near homogeneity none of these other recombinant forms of PSA cleaved Tau. Further, Tau-cleaving activity and aminopeptidase activities derived from the Sf9 cell expression system were separable by molecular sieve chromatography. When tested in a cellular context we again failed to see a PSA dependent cleavage of Tau. A commercial preparation of a related aminopeptidase, aminopeptidase N, also exhibited Tau cleaving activity, but this activity could also be separated from aminopeptidase activity. CONCLUSION: It is concluded that PSA does not directly cleave Tau.