RESUMO
The result of a deprivation of oxygen and glucose to the brain, hypoxic-ischemic encephalopathy (HIE), remains the most common cause of death and disability in human neonates globally and is mediated by glutamate toxicity and inflammation. We have previously shown that the enzyme glutamate carboxypeptidase (GCPII) is overexpressed in activated microglia in the presence of inflammation in fetal/newborn rabbit brain. We assessed the therapeutic utility of a GCPII enzyme inhibitor called 2-(3-Mercaptopropyl) pentanedioic acid (2MPPA) attached to a dendrimer (D-2MPPA), in order to target activated microglia in an experimental neonatal hypoxia-ischemia (HI) model using superoxide dismutase transgenic (SOD) mice that are often more injured after hypoxia-ischemia than wildtype animals. SOD overexpressing and wild type (WT) mice underwent permanent ligation of the left common carotid artery followed by 50 min of asphyxiation (10% O2) to induce HI injury on postnatal day 9 (P9). Cy5-labeled dendrimers were administered to the mice at 6 h, 24 h or 72 h after HI and brains were evaluated by immunofluorescence analysis 24 h after the injection to visualize microglial localization and uptake over time. Expression of GCPII enzyme was analyzed in microglia 24 h after the HI injury. The expression of pro- and anti-inflammatory cytokines were analyzed 24 h and 72 h post-HI. Brain damage was analyzed histologically 7 days post-HI in the three randomly assigned groups: control (C); hypoxic-ischemic (HI); and HI mice who received a single dose of D-2MPPA 6 h post-HI (HI+D-2MPPA). First, we found that GCPII was overexpressed in activated microglia 24 h after HI in the SOD overexpressing mice. Also, there was an increase in microglial activation 24 h after HI in the ipsilateral hippocampus which was most visible in the SOD+HI group. Dendrimers were mostly taken up by microglia by 24 h post-HI; uptake was more prominent in the SOD+HI mice than in the WT+HI. The inflammatory profile showed significant increase in expression of KC/GRO following injury in SOD mice compared to WT at 24 and 72 h. A greater and significant decrease in KC/GRO was seen in the SOD mice following treatment with D-2MPPA. Seven days after HI, D-2MPPA treatment decreased brain injury in the SOD+HI group, but not in WT+HI. This reduced damage was mainly seen in hippocampus and cortex. Our data indicate that the best time point to administer D-2MPPA is 6 h post-HI in order to suppress the expression of GCPII by 24 h after the damage since dendrimer localization in microglia is seen as early as 6 h with the peak of GCPII upregulation in activated microglia seen at 24 h post-HI. Ultimately, treatment with D-2MPPA at 6 h post-HI leads to a decrease in inflammatory profiles by 24 h and reduction in brain injury in the SOD overexpressing mice.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutaratos/farmacologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores , Compostos de Sulfidrila/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/patologia , Dendrímeros/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Hipóxia-Isquemia Encefálica/genética , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
While a lot is known about classical, anterograde neurotransmission, less is known about the mechanisms and molecules involved in retrograde neurotransmission. Our hypothesis is that N-acetylaspartylglutamate (NAAG), the most abundant dipeptide in the brain, may act as a retrograde transmitter in the brain. NAAG was predominantly localized in dendritic compartments of glutamatergic synapses in the intact hippocampus, where it was present in close proximity to synaptic-like vesicles. In acute hippocampal slices, NAAG was depleted from postsynaptic dendritic elements during neuronal stimulation induced by depolarizing concentrations of potassium or by exposure to glutamate receptor (GluR) agonists. The depletion was completely blocked by botulinum toxin B and strictly dependent on extracellular calcium, indicating exocytotic release. In contrast, there were low levels of NAAG and no effect by depolarization or GluR agonists in presynaptic glutamatergic terminals or GABAergic pre- and postsynaptic elements. Together these data suggest a possible role for NAAG as a retrograde signaling molecule at glutamatergic synapses via exocytotic release.
Assuntos
Dipeptídeos/farmacologia , Exocitose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Exocitose/fisiologia , Hipocampo/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos Wistar , Vesículas Sinápticas/efeitos dos fármacosRESUMO
Could immunization against cocaine be a viable prophylaxis for cocaine addiction?
Assuntos
Cocaína/imunologia , Imunização , Transtornos Relacionados ao Uso de Substâncias/imunologia , Transtornos Relacionados ao Uso de Substâncias/prevenção & controle , Animais , Cocaína/química , Humanos , Modelos MolecularesRESUMO
We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.
Assuntos
Isquemia Encefálica/prevenção & controle , Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fármacos Neuroprotetores/farmacologia , Compostos Organofosforados/farmacologia , Animais , Isquemia Encefálica/metabolismo , Carboxipeptidases/metabolismo , Técnicas de Cultura , Dipeptídeos/metabolismo , Modelos Animais de Doenças , Tolerância a Medicamentos , Glutamato Carboxipeptidase II , Ácido Glutâmico/metabolismo , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismoRESUMO
The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.
Assuntos
Antineoplásicos/toxicidade , Neuropatia Ciática , Animais , Modelos Animais de Doenças , Epotilonas/toxicidade , Feminino , Furanos/toxicidade , Filamentos Intermediários/metabolismo , Cetonas/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Paclitaxel/toxicidade , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Proteínas S100/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Neuropatia Ciática/induzido quimicamente , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
Nearly two decades ago, Joe Coyle published a single-authored review with the provocative title, The Nagging Question of the Function of N-Acetylaspartylglutamate (Coyle, 1997). In this review, Coyle documented NAAG's localization to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neurons, Ca(2+)-dependent release, mGlu3 receptor agonist and NMDA receptor antagonist activity, and cleavage by the glial enzyme glutamate carboxypeptidase II (GCPII). However, at the time of his review, NAAG's physiological function as a neurotransmitter remained elusive. Ironically his review was published months following the discovery of the first potent and selective GCPII inhibitor, 2-(phosphonomethyl)pentanedioc acid (2-PMPA) (Jackson et al., 1996). Over the ensuing decades, over a dozen independent laboratories used 2-PMPA and other GCPII inhibitors to elucidate two distinct neurotransmitter functions for NAAG. Under basal conditions, when GCPII activity is relatively low, intact NAAG dampens synaptic activity via presynaptic mGlu3 receptor activation and NMDA receptor blockade. However, under stimulated conditions, NAAG release and GCPII activity are enhanced resulting in excess glutamate generation, activating NMDA and other glutamate receptors, often pathologically. Diverse classes of GCPII inhibitors have been synthesized and shown to increase NAAG, decrease glutamate, and provide robust efficacy in many disease models wherein abnormal glutamatergic transmission is presumed pathogenic. In addition, over the past 20 years, basic questions regarding NAAG's synthesis, packaging into vesicles, and receptor selectivity profile have been eloquently elucidated. The purpose of this chapter is to summarize these advances and the promise of regulating NAAG metabolism through GCPII inhibition as a therapeutic strategy.
Assuntos
Dipeptídeos/metabolismo , Glutamato Carboxipeptidase II/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Animais , Antígenos de Superfície , Humanos , Neuroglia/metabolismo , Neurotransmissores/metabolismoRESUMO
Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3'-OH kinase (PI3K)/Akt cascades. Mx1-Cre, Kras(D12) and Mx1-Cre, Nf1(flox/)(-) mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, Kras(D12) mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.
Assuntos
Compostos Heterocíclicos com 3 Anéis/farmacologia , Síndromes Mielodisplásicas/metabolismo , Transtornos Mieloproliferativos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas ras/genética , Animais , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Indazóis , Leucemia Mielomonocítica Crônica , Leucemia Mielomonocítica Juvenil , Sistema de Sinalização das MAP Quinases , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Transtornos Mieloproliferativos/tratamento farmacológico , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , SulfonamidasRESUMO
BACKGROUND: Schizophrenia has been hypothesized to be caused by a hypofunction of glutamatergic neurons. Findings of reduced concentrations of glutamate in the cerebrospinal fluid of patients with schizophrenia and the ability of glutamate-receptor antagonists to cause psychotic symptoms lend support to this hypothesis. N-acetylaspartylglutamate (NAAG), a neuropeptide that is highly concentrated in glutamatergic neurons, antagonizes the effects of glutamate at N-methyl-D-aspartate receptors. Moreover, NAAG is cleaved to glutamate and N-acetylaspartate by a specific peptidase, N-acetyl-alpha-linked acidic dipeptidase (NAALADase). To test the glutamatergic hypothesis of schizophrenia, we studied the NAAG-related glutamatergic variables in postmortem brains from patients with schizophrenia, neuroleptic-treated controls, and normal individuals, with particular emphasis on the prefrontal cortex and hippocampus. METHOD: Different regions of frozen brain tissue from three different groups (patients with schizophrenia, neuroleptic-treated controls, and normal controls) were assayed to determine levels of NAAG, N-acetylaspartate, NAALADase, and several amino acids, including aspartate and glutamate. RESULTS: Our study demonstrates alterations in brain levels of aspartate, glutamate, and NAAG and in NAALADase activity. Levels of NAAG were increased and NAALADase activity and glutamate levels were decreased in the schizophrenic brains. Notably, the changes in NAAG level and NAALADase activity in schizophrenic brains were more selective than those for aspartate and glutamate. In neuroleptic-treated control brains, levels of aspartate, glutamate, and glycine were found to be increased. CONCLUSIONS: The changes in levels of aspartate, glutamate, NAAG, and NAALADase are prominent in the prefrontal and hippocampal regions, where previous neuropathological studies of schizophrenic brains demonstrate consistent changes. These findings support the hypothesis that schizophrenia results from a hypofunction of certain glutamatergic neuronal systems. They also suggest that the therapeutic efficacy of neuroleptics may be related to increased glutamatergic activity.
Assuntos
Antígenos de Superfície , Encéfalo/metabolismo , Glutamatos/metabolismo , Receptores de Glutamato/metabolismo , Esquizofrenia/metabolismo , Adulto , Idoso , Encéfalo/enzimologia , Encéfalo/fisiologia , Dipeptidases/metabolismo , Dipeptidases/fisiologia , Dipeptídeos/metabolismo , Dipeptídeos/fisiologia , Feminino , Glutamato Carboxipeptidase II , Hipocampo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/metabolismo , Receptores de Glutamato/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/fisiopatologiaRESUMO
A series of trihexyphenidyl (THP) analogs were used to search for a derivative that could serve as a cocaine inhibitor. A compound that blocks binding of the cocaine analog carboxyfluorotropane (CFT), allows dopamine uptake and exhibits low side effects could serve as a good candidate for that purpose. All analogs were tested for the extent to which they inhibit CFT binding, dopamine uptake and n-methyl scopolamine (NMS) binding. Several structure-function relationships emerged. Methylation/halogenation of THP's benzene ring enhanced the compound's ability to block CFT binding in comparison to its ability to block dopamine uptake (5a-e). Replacement of the cyclohexyl ring with a benzene ring tended to create compounds that had lower affinities to the dopamine transporter (7b compared to THP, 7d compared to 5h, 7c compared to 8c) and modification of THP's piperidine ring tended to enhance affinity to the dopamine transporter (5f-h, 8a, 8c). One analog (5f) that showed little muscarinic activity indicating that it would probably have few side effects was investigated for its effects as an in vivo cocaine inhibitor. However, it showed few antagonistic effects in vivo. Nevertheless, this work greatly elucidates the structure-function relationships required for potential cocaine inhibitors and so lays out promising directions for future research.
Assuntos
Cocaína/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Triexifenidil/análogos & derivados , Triexifenidil/farmacologia , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Triexifenidil/químicaRESUMO
N-acetylated alpha-linked acidic dipeptidase (NAALADase) is a membrane bound enzyme that cleaves glutamate from the endogenous neuropeptide N-acetyl-aspartyl-glutamate (NAAG). We report the immunocytochemical localization of NAALADase in rat brain and kidney by using specific anti-NAALADase antiserum. NAALADase-immunoreactivity (NAALADase-IR) was widely distributed, abundant in neuropil, absent from neuronal cytoplasm, and displayed regional heterogeneity. Staining was selectively enriched in several structures previously reported to contain NAAG-immunoreactivity (NAAG-IR) including the amygdala, caudate-putamen, central gray, dorsal raphe, globus pallidus, hippocampus, hypothalamus, locus coerulus, medial and lateral geniculate, olfactory bulb, periaqueductal gray, solitary nucleus, spinal trigeminal nucleus, substantia nigra, superior colliculus, and thalamus. Staining within these structures was enriched in neuropil; no intracellular staining was detected, even after colchicine treatment. In addition, NAALADase-IR was observed in some NAAG-containing fiber tracts including the corpus callosum, fornix, habenular commissure, solitary tract, stria medularis, and stria terminalis. The co-localization of NAALADase-IR and NAAG-IR support the hypothesis that NAALADase is responsible for the catabolism of NAAG in vivo. NAALADase-IR was also detected in brain regions that, to date, have not revealed NAAG-IR, including the bed nucleus of the stria terminalis and the median eminence. In addition, NAALADase-IR was detected in the rat kidney cortex, specifically in the brush border of the proximal convoluted tubules. The observation that NAALADase-IR was more widespread than NAAG-IR suggests that NAALADase may also be involved in the catabolism of other structurally related neural and renal peptides.
Assuntos
Encéfalo/enzimologia , Dipeptidases/análise , Túbulos Renais Proximais/enzimologia , Animais , Dipeptídeos/metabolismo , Glutamato Carboxipeptidase II , Masculino , Microvilosidades/enzimologia , Proteínas do Tecido Nervoso/análise , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Medula Espinal/enzimologiaRESUMO
Excessive glutamatergic transmission is thought to be responsible for the injury observed in a variety of neurological disorders such as stroke. N-acetylaspartylglutamate (NAAG), a major peptidic component of the brain, has been suggested to serve as a potential storage form of glutamate. N-acetylated-a-linked acidic dipeptidase (NAALADase, EC 3.4.17.21) is responsible for the hydrolysis of NAAG into N-acetylaspartate (NAA) and glutamate. If NAAG is a storage form of glutamate, then inhibition of NAALADase should be neuroprotective in diseases in which excess glutamatergic transmission is detrimental. In addition, NAAG has been demonstrated to be an agonist at group II metabotropic glutamate receptors and functions as a mixed agonist/antagonist at N-methyl-D-aspartate receptors. Therefore, inhibition of NAALADase would also function to increase NAAG levels which, in turn, should provide neuroprotection via the interaction of NAAG with these receptors. Recently, potent and selective inhibitors of the enzyme have been designed and subsequently used to demonstrate that inhibition of NAALADase is neuroprotective in animal models of neurodegeneration. As such, NAALADase inhibition represents a novel method of regulating extracellular glutamate levels and provides a new avenue for the treatment of neurological disorders.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Ácidos Carboxílicos/química , Dipeptídeos/metabolismo , Inibidores Enzimáticos/química , Glutamato Carboxipeptidase II , Ácido Glutâmico/metabolismo , Isquemia/tratamento farmacológico , Fármacos Neuroprotetores/química , Ácidos Fosfínicos/química , Fósforo/química , Receptores de Glutamato Metabotrópico/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of substituted phosphonate derivatives were designed and synthesized in order to study the ability of these compounds to inhibit the neuropeptidase N-acetylated alpha-linked acidic dipeptidase (NAALADase). The molecules were shown to act as inhibitors of the enzyme, with the most potent (compound 3) having a Ki of 0.275 nM. The potency of this compound is more than 1000 times greater than that of previously reported inhibitors of the enzyme. NAALADase is responsible for the catabolism of the abundant neuropeptide N-acetyl-aspartylglutamate (NAAG) into N-acetylaspartate and glutamate. NAAG has been proposed to be a neurotransmitter at a subpopulation of glutamate receptors; alternatively, NAAG has been suggested to act as a storage form of synaptic glutamate. As a result, inhibition of NAALADase may show utility as a therapeutic intervention in diseases in which altered levels of glutamate are thought to be involved.
Assuntos
Dipeptidases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Acetilação , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/metabolismo , Dipeptidases/química , Dipeptidases/metabolismo , Dipeptídeos/metabolismo , Inibidores Enzimáticos/farmacologia , Glutamato Carboxipeptidase II , Concentração de Íons de Hidrogênio , Neuropeptídeos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
A novel series of phosphinic acid based inhibitors of the neuropeptidase NAALADase are described in this work. This series of compounds is the most potent series of inhibitors of the enzyme described to date. In addition, we have shown that these compounds are protective in animal models of neurodegeneration. Compound 34 significantly prevented neurodegeneration in a middle cerebral artery occlusion model of cerebral ischemia. In addition, in the chronic constrictive model of neuropathic pain, compound 34 significantly attenuated the hypersensitivity observed with saline-treated animals. These data suggest that NAALADase inhibition may provide a new approach for the treatment of both neurodegenerative disorders and peripheral neuropathies.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Fármacos Neuroprotetores/síntese química , Ácidos Fosfínicos/síntese química , Animais , Arteriopatias Oclusivas/prevenção & controle , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glutamato Carboxipeptidase II , Artéria Cerebral Média , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Medição da Dor , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/fisiopatologia , Ácidos Fosfínicos/química , Ácidos Fosfínicos/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Crayfish nerve fibers incubated with radiolabeled glutamate or glutamine accumulate these substrates and synthesize radioactive N-acetylaspartylglutamate (NAAG). Upon stimulation of the medial giant nerve fiber, NAAG is the primary radioactive metabolite released. Since NAAG activates a glial hyperpolarization comparable to that initiated by glutamate or axonal stimulation through the same receptor, we have proposed that it is the likely mediator of interactions between the medial giant axon and its periaxonal glia. This manuscript reports investigations of possible mechanisms for termination of NAAG-signaling activity. N-acetylaspartyl-[(3)H]glutamate was not accumulated from the bath saline by unstimulated crayfish giant axons or their associated glia during a 30-min incubation. Stimulation of the central nerve cord at 50 Hz during the last minute of the incubation dramatically increased the levels of radiolabeled glutamate, NAAG, and glutamine in the medial giant axon and its associated glia. These results indicate that stimulation-sensitive peptide hydrolysis and metabolic recycling of the radiolabeled glutamate occurred. There was a beta-NAAG-, quisqualate- and 2-(phosphonomethyl)-pentanedioic acid-inhibitable glutamate carboxypeptidase II activity in the membrane fraction of central nerve fibers, but not in axonal or glial cytoplasmic fractions. Inactivation of this enzyme by 2-(phosphonomethyl)-pentanedioic acid or inhibition of N-methyl-D-aspartate (NMDA) receptors by MK801 reduced the glial hyperpolarization activated by high-frequency stimulation. These results indicate that axon-to-glia signaling is terminated by NAAG hydrolysis and that the glutamate formed contributes to the glial electrical response in part via activation of NMDA receptors. Both NAAG release and an increase in glutamate carboxypeptidase II activity appear to be induced by nerve stimulation.
Assuntos
Dipeptídeos/farmacocinética , Fibras Nervosas/metabolismo , Neuroglia/fisiologia , Transdução de Sinais/fisiologia , Animais , Astacoidea , Carboxipeptidases/metabolismo , Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Citoplasma/metabolismo , Maleato de Dizocilpina/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/metabolismo , Glutamato Carboxipeptidase II , Neuroglia/citologia , Compostos Organofosforados/farmacologia , TrítioRESUMO
N-acetylaspartylglutamate (NAAG) is an acidic dipeptide found in high concentration throughout the rat central nervous system. NAAG has been proposed as a neurotransmitter/modulator in some excitatory glutamatergic pathways where it is released by a Ca(2+)-dependent process with neuronal activity. Previous immunocytochemical studies have revealed few neurons exhibiting NAAG-like immunoreactivity (LI) in the forebrain, especially in putative glutamatergic neurons. In this study, we present a detailed map of NAAG-LI in rat forebrain utilizing a modified fixation technique that markedly enhances sensitivity. NAAG-LI is located in most of the putative glutamatergic pathways in the forebrain including pyramidal neurons in motor and sensory cortices and the hippocampal formation. Co-localization of NAAG-LI to cholinergic systems of the forebrain was quite extensive with the exception of the striatal local circuit neurons. A noteworthy feature of NAAG-LI-positive neuronal groups is that they were often configured in hierarchical relationships. For example, the pyramidal neurons of the motor cortex and the motor neurons of the brainstem and and spinal cord expressed NAAG-LI; also, several inter-related components of the limbic system stained for NAAG-LI. Taken together, these findings indicate that NAAG is a neuropeptide localized to subpopulations of neurons throughout forebrain as well as in brainstem and spinal cord.
Assuntos
Dipeptídeos/análise , Glutamatos/fisiologia , Neuropeptídeos/análise , Neurotransmissores/fisiologia , Prosencéfalo/química , Animais , Diencéfalo/química , Ácido Glutâmico , Imuno-Histoquímica , Sistema Límbico/química , Masculino , Neurotransmissores/análise , Ratos , Ratos Sprague-Dawley , Telencéfalo/química , Tálamo/químicaRESUMO
RATIONALE: Long-term individual housing increases aggressive behavior in mice, a condition termed isolation-induced aggression; this aggressiveness is reduced by some antidepressants and anxiolytics. NMDA antagonists also inhibit isolation-induced aggression in mice. The enzyme N-acetylated-alpha-linked acidic dipeptidase (NAALADase) hydrolyzes the neurotransmitter N-acetylaspartylglutamate (NAAG) to form glutamate and N-acetylaspartate; NAAG acts as a partial NMDA agonist as well as a full agonist at the presynaptic metabotropic glutamate receptor 3 (mGluR3), where it acts to reduce glutamate release. OBJECTIVE: We postulated that NAALADase inhibition would reduce isolation-induced aggression in mice. METHODS: We tested whether acute exposure to the NAALADase inhibitor 2-[[hydroxy[2,3,4,5,6-pentafluorophenyl)methyl]phosphinyl]methyl] pentanedioic acid (GPI-5232), administered 30 min prior to a social interaction test, would inhibit aggressive behavior in SJL mice that had been individually housed long term. RESULTS: Administration of GPI-5232 (30 mg/kg, IP) inhibited initiation of aggressive behavior, indicated by greater latencies to display tail-rattling, attack and biting, and by fewer mice initiating aggressive behavior, compared to mice that received vehicle. In addition, GPI-5232 treated mice had fewer tail-rattling responses to a non-aggressive conspecific. CONCLUSIONS: The effectiveness of GPI-5232 in this animal model suggests that NAALADase inhibition may be a novel therapeutic approach to reduce or inhibit heightened aggressiveness, and possibly to treat aggressive behavior associated with psychiatric disorders.
Assuntos
Agressão/efeitos dos fármacos , Agressão/psicologia , Inibidores Enzimáticos/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Isolamento Social/psicologia , Agressão/fisiologia , Animais , Glutamato Carboxipeptidase II/metabolismo , Glutaratos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Hereditary canine spinal muscular atrophy (HCSMA) is a lower motor neuron disease found in Brittany Spaniels that shares clinical and pathological features with human amyotrophic lateral sclerosis (ALS). Since acidic excitatory amino acids and the neuropeptide N-acetyl-aspartyl-glutamate (NAAG) are reduced in spinal cord and cerebral cortex in ALS, the levels of these substances were measured in nervous tissue in Brittany Spaniels heterozygous and homozygous for HCSMA. Significant reductions in the levels of endogenous aspartate, glutamate, N-acetylaspartate (NAA), and NAAG were found in the spinal cord in homozygous but not heterozygous HCSMA. In contrast, the activity of N-acetylated-alpha-linked-amino dipeptidase (NAALADase), an enzyme that cleaves NAAG into NAA and Glu, was significantly increased. None of these parameters was affected in the motor cortex or occipital cortex. Since NAA and NAAG are highly concentrated in motoneurons, they may play a role in the pathogenesis of motor neuron disease.
Assuntos
Aminoácidos/metabolismo , Dipeptídeos/metabolismo , Doenças do Cão/metabolismo , Atrofia Muscular Espinal/metabolismo , Neuropeptídeos/metabolismo , Aminoácidos/sangue , Aminoácidos/líquido cefalorraquidiano , Animais , Biomarcadores , Química Encefálica/fisiologia , Dipeptídeos/sangue , Dipeptídeos/líquido cefalorraquidiano , Doenças do Cão/genética , Cães , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/veterinária , Neuropeptídeos/sangue , Neuropeptídeos/líquido cefalorraquidiano , Medula Espinal/metabolismoRESUMO
N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a membrane-bound peptidase which hydrolyzes the endogenous neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and the excitatory amino acid, glutamate (Glu). Although there is evidence that NAAG might be a neurotransmitter, this dipeptide could also function as a precursor form of Glu, which is liberated by the dipeptidase. We found that the activity of this NAAG hydrolyzing enzyme in genetically epilepsy-prone rats was 11-26% greater than control in brain regions, including the amygdala, hippocampus and cerebellum, as well as the pyriform, entorhinal and frontal cortices. This is consistent with possible increased availability of Glu in certain CNS synapses in these rats, which are reported to have increased susceptibility to audiogenically, electrically and chemically induced convulsions.
Assuntos
Encéfalo/enzimologia , Dipeptidases/metabolismo , Dipeptídeos/metabolismo , Epilepsia/enzimologia , Neuropeptídeos/metabolismo , Animais , Membrana Celular/enzimologia , Epilepsia/genética , Glutamato Carboxipeptidase II , Glutamatos/metabolismo , Ácido Glutâmico , Masculino , Especificidade de Órgãos , Ratos , Ratos Mutantes , Valores de ReferênciaRESUMO
Folates have been shown to be neurotoxic and convulsive. Endogenously, folates exist in the brain in a polyglutamated form with 1-7 terminal glutamates (approx. 1 microM). The brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase) has been shown to remove sequentially the gamma-linked glutamates from folic acid polyglutamates. We report that, at high concentrations (300 microM-30 mM), a folic acid hexaglutamate analog is dose-dependently toxic to dissociated rat cortical cultures and that this toxicity is reversed by 2-PMPA, a potent and selective NAALADase inhibitor. These data suggest a new mechanism for folic acid toxicity.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Metotrexato/análogos & derivados , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Compostos Organofosforados/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Benzoatos/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Desoxiglucose/farmacologia , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feto , Glutamato Carboxipeptidase II , Glicina/análogos & derivados , Glicina/farmacologia , Metotrexato/antagonistas & inibidores , Metotrexato/toxicidade , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/fisiologia , Cianeto de Potássio/farmacologia , Pirrolidinas/farmacologia , RatosRESUMO
2-Phosphonomethyl pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase II (NAALADase), and has shown robust neuroprotective activity in both in vitro and in vivo models of ischemia. In the brain, glutamate carboxypeptidase II (GCPII) (EC3.4.17.21) hydrolyzes the neuropeptide N-acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate. We report the development and characterization of a [(3)H]2-PMPA binding assay. [(3)H]2-PMPA binding was dependent on protein concentration, saturable, and displaceable. The association (k(on)) and dissociation (k(off)) rate constants were 3x10(6) M(-1) s(-1) and 0.01 s(-1), respectively. The dissociation equilibrium constant (K(d)) determined from the ratio of the rate constants (K(d)=k(off)/k(on)) was 1 nM. Scatchard analysis revealed one binding site with K(d)=2 nM and B(max)=0.7 pmol/mg. Binding exhibited similar pharmacological properties to GCPII enzyme activity, including chloride dependency, cobalt stimulation and inhibition by phosphate and quisqualate. The binding of [(3)H]2-PMPA also showed tissue specificity in that tissues previously reported to be devoid of GCPII enzymatic activity were devoid of [(3)H]2-PMPA binding. [(3)H]2-PMPA binding represents an additional probe for the study of GCPII activity, and may be useful as a high throughput screening assay.