Constipation is a serious public health problem that afflicts thousands of patients worldwide. It is believed that with the modern lifestyle, followed by constant stress and inadequate eating habits, the incidence of constipation will increase considerably in coming decades. Constipation is caused by abnormal functioning of the digestive tract which is not yet fully elucidated. It is known that the enteric nervous system is responsible for sensory and motor functions of the digestive tract, which makes it work in perfect sync and perform peristalsis, promoting proper transit of the bolus and subsequently fecal mass. The vast majority of pathologies afflicting the gastrointestinal tract are originated from disturb in specific neurons in the enteric nervous system and probably the same happens with constipation. Hence, the objective of this study was, by immunohistochemistry, to characterize and compare the expression of several neuropeptides of the enteric nervous system in patients with constipation and individuals not constipated. The results showed that among the various types of neurons studied, constipated patients have fewer neurons expressing calretinin and choline acetyltransferase, characteristic neuropeptides from afferent neurons and excitatory neurons engines. We believe that these results can help in future treatment techniques and prevention of constipation. / A constipação intestinal é um problema grave de saúde pública que aflige milhares de pacientes em todo o mundo. Acredita-se que, com o estilo de vida moderno, seguido de constante estresse e inadequação dos hábitos alimentares, a incidência da constipação intestinal aumente consideravelmente nas próximas décadas. A constipação é causada por alterações no funcionamento do trato digestório que ainda não estão completamente elucidadas. Sabe-se que o sistema nervoso entérico é responsável pelas funções sensitivas e motoras do trato digestivo, o que confere a este funcionar sob perfeita sincronia e realizar a peristalse, promovendo o trânsito adequado do bolo alimentar e posteriormente do bolo fecal. A grande maioria das patologias que afligem o trato gastrointestinal são originadas de distúrbios em neurônios específicos do sistema nervoso entérico e, provavelmente, o mesmo ocorre com a constipação intestinal. Diante disto, o objetivo deste trabalho foi, através da técnica de imunohistoquímica, caracterizar e comparar a expressão dos diversos neuropeptídios do sistema nervoso entérico em pacientes portadores de constipação intestinal e indivíduos não constipados. Os resultados demonstraram que, dentre os vários tipos de neurônios estudados, os pacientes constipados apresentam uma menor quantidade de neurônios que expressam calretinina e colina-acetiltransferase, neuropeptídios característicos de neurônios aferentes e de neurônios motores excitatórios. Acreditamos que esses resultados possam colaborar com futuras técnicas de tratamento e com a prevenção da constipação intestinal. / Mestre em Ciências da Saúde

Introdução: A urocortina 3 (UCN 3) é um neuropeptídeo pertencente a família CRF, com seletividade de ligação a receptor CRF2. Em roedores as células UCN 3 se localizam principalmente em hipotálamo e amígdala, mas o mapeamento não foi realizado em modelo de primata não-humano. Objetivo: realizar o mapeamento da distribuição da UCN 3 no sistema nervoso central na espécie Cebus apella. Material e métodos: cortes de encéfalo de animais machos foram submetidos aos métodos de imuno-histoquímica e hibridização in situ para UCN 3. Séries adjacentes foram coradas pelo método de Nissl e hematoxilina-eosina. Resultados: Células UCN 3 se localizam principalmente em regiões hipotalâmicas, amigdalóides e límbicas. Há colocalização de UCN 3/CRF no núcleo paraventricular do hipotálamo e UCN 3/insulina em células <font face=\"Symbol\">b do pâncreas. Conclusão: a distribuição de UCN 3 em primata não-humano é semelhante à de roedores. / Introduction: Urocortin 3 (UCN 3) is a neuropeptide with 38-aa and member of the CRF peptide family, it is a selective agonist for the CRF2 receptor. UCN 3 cells in rodents showed containing- neurons found mainly in hypothalamic and amygdaloid regions. However, such mapping was not done in a non-human primate model. Objective: study the UCN 3 distribution in the brain of a monkey. Material and methods: frontal sections (40<font face=\"Symbol\">mm) were subjected to immunohistochemistry technique and in situ hybridization, Nissl and Hematoxylin-eosin staining. Results: UCN 3 cells were found mainly in the amygdaloid, limbic and hypothalamic regions. Double-labeled cells (CRF/UCN 3) were found in the PaMD and, in <font face=\"Symbol\">b cells (UCN 3/insulin) of pancreas. Conclusion: the distribution of UCN 3 in non-human primate is similar to the rodents distribution.

As teneurinas (TENs) são de proteínas de transmembrana com significante expressão no sistema nervoso central (SNC). Foi verificado que o último éxon dos genes das TENs codifica um peptídeo com elevada identidade ao fator liberador de corticotropina (CRF) sendo este denominado de peptídeo associado ao terminal carboxila da teneurina (TCAP-1 a 4). Estudos mostram que TCAP-1 controla o comportamento emocional possivelmente por modular as ações mediadas pelo (CRF). A distribuição do TCAP-1 no SNC de primatas poderia subsidiar os seus efeitos funcionais e as possíveis aplicações terapêuticas. O presente trabalho analisou a imunorreatividade e a expressão do RNA mensageiro do TCAP-1 no encéfalo de primatas não-humanos. Cortes frontais do SNC de macacos da espécie Cebus apella (n=3) foram selecionados e utilizados nas técnicas de hibridização in situ e de imuno-histoquímica. Neurônios imunorreativos e que expressam o RNAm para TCAP-1 foram encontrados principalmente em áreas que estão direta ou indiretamente envolvidas na modulação da resposta ao estresse e ansiedade. / Teneurins are transmembrane proteins expressed mainly in the central nervous system (CNS). The last exon of the teneurins exhibits a peptide sequence with high homology to corticotrophin-releasing factor (CRF), named teneurin c-terminal associated peptide (TCAP-1 to 4). TCAP-1 effectively modulates stress induced by CRF. Studies have pointed tha TCAP-1 could have important therapeutic applications in stress disorders. The analysis of TCAP-1 distribution in the primate brain could also give anatomical support to the understanding of its functional effects and possible therapeutic use. Thus, the present study focused on the distribution of neurons exhibiting immunoreactivity and mRNA expression to TCAP-1 in the monkey brain (Cebus apella). Frontal brain sections of three young male Cebus apella monkeys were submitted to immunohistochemistry and in situ hybridization. Immunohistochemistry and in situ hybridization results showed that TCAP-1 is preserved in primate brain, mainly in areas directly or indirectly involved in the modulation of stress and anxiety.

Os transtornos neuropsiquiátricos incluem-se entre as patologias de alta incidência, difícil identificação e prognósticos variados. Dentre eles se destaca a esquizofrenia, uma doença funcional do cérebro caracterizada essencialmente por uma fragmentação da estrutura básica dos processos de pensamento, acompanhada pela dificuldade em estabelecer a distinção entre experiências internas e externas. O presente estudo visa à investigação do risco cardíaco decorrente do ganho de peso causado pelo uso do antipsicótico olanzapina em pacientes esquizofrênicos, bem como a avaliação dos níveis dos neuropeptídeos relacionados ao balanço energético, a fim de estabelecer o mecanismo de ação responsável por este ganho de peso. Como a identificação conclusiva dos fatores etiológicos ou patogênicos dos transtornos neuropsiquiátricos permanece desconhecida, o trabalho visa ainda à avaliação dos níveis plasmáticos de alguns aminoácidos neurotransmissores, correlacionando-os a estes transtornos. Para a avaliação do risco cardíaco e do possível mecanismo do ganho de peso, um grupo de 30 pacientes com diagnóstico de esquizofrenia em início de terapia com a olanzapina foi submetido a avaliações antropométricas, bioquímicas e determinação plasmática de grelina, leptina, neuropeptídeo NPY e polipeptídeo YY durante 12 meses. A investigação da correlação entre transtornos neuropsiquiátricos e aminoácidos, foi realizada em outros 150 indivíduos subdivididos em cinco grupos de 30 pessoas, pacientes com diagnóstico de esquizofrenia, epilepsia, depressão e transtorno afetivo bipolar, os quais foram comparados a um grupo controle. Os resultados obtidos referentes ao estudo do risco cardíaco foram demonstrados através do aumento significativo do peso, índice de massa corporal e circunferências de cintura e quadril. Em relação aos parâmetros bioquímicos, verificaram-se alterações clinicamente significativas nos níveis de colesterol, triglicérides, LDL-colesterol, glicose, insulina e cortisol. Quanto aos neuropeptídeos, observou-se aumento significativo na grelina e neuropeptídeo NPY. Os níveis plasmáticos dos aminoácidos não essenciais, glutamato, aspartato, serina, glicina e arginina, por sua vez, mostraram-se significativamente alterados nas patologias neuropsiquiátricas quando comparados ao grupo controle, sendo que o glutamato apresentou incremento altamente significante em todas as patologias neuropsiquiátricas estudadas, evidenciando a íntima correlação entre este aminoácido e os transtornos supracitados, bem como a hipótese da hiperfunção glutamatérgica em nível periférico, isto é, valores plasmáticos. Neste contexto, evidenciou-se que a terapia com a olanzapina aumenta o risco cardíaco em decorrência da maior liberação dos hormônios orexígenos, podendo comprometer a qualidade de vida destes pacientes por favorecer a síndrome metabólica. Dessa forma, o estudo contribui para elucidação deste marcante efeito adverso da olanzapina que é o ganho de peso. Evidenciou-se, ainda, que a determinação dos aminoácidos plasmáticos, aliada a outras técnicas de avaliação em desenvolvimento e pesquisa, poderão servir como marcadores biológicos para os trantornos neuropsiquiátricos, bem como de medidas preventivas e no estabelecimento de uma nova perspectiva para alvos de futuros fármacos adjuvantes à terapia neuropsiquiátrica. / Neuropsychiatric disorders are among diseases with high incidence, difficult to identify and with varied predictions. Among them stands out schizophrenia, a brain functional disease essentially characterized by fragmentation of the basic structure of thinking processes, accompanied by difficulty to distinguish between internal and external experiences. The present study aims to investigate cardiac risk in schizophrenic patients due to weight gain caused by the use of antipsychotic olanzapine, as well as evaluating the levels of neuropeptides related to energy balance in order to establish the mechanism of action responsible for such weight gain. As the conclusive identification of etiological or pathogenic factors of neuropsychiatric disorders remains unknown, the work also aims to assess the plasma levels of some neurotransmitter amino acids, correlating them with these disorders. For the evaluation of cardiac risk and the possible mechanism of weight gain, a group of 30 patients diagnosed with schizophrenia and at the start of olanzapine therapy underwent anthropometric evaluations and biochemical quantification of serum ghrelin, leptin, and neuropeptide NPY polypeptide YY 12 months. The investigation of the correlation between neuropsychiatric disorders and amino acids was performed in other 150 individuals divided into five groups of 30 persons, patients with schizophrenia, epilepsy, depression and bipolar disorder, which were compared to a control group. The results obtained for the study of cardiac risk were demonstrated by the significant increase in weight, body mass index and waist and hip circumferences. Regarding biochemical parameters, there were no clinically significant changes in cholesterol, triglycerides, LDL-cholesterol, glucose, insulin and cortisol. Concerning neuropeptides, we could observe a significant increase in ghrelin and neuropeptide NPY. Plasma levels of nonessential amino acids, glutamate, aspartate, serine, glycine and arginine, in turn, were significantly altered in neuropsychiatric disorders. Glutamate showed a highly significant increase in all studied neuropsychiatric disorders, showing the close correlation between this amino acid and the aforementioned disorders, as well as the glutamatergic hyperfunction hypothesis at peripheral level, that is, plasma levels. In this context, it was observed that therapy with olanzapine increases cardiac risk due to augmented release of orexigenic hormones, which can compromise the quality of life of patients by promoting metabolic syndrome. Thus, it contributes to elucidation of this striking side effect of olanzapine which is the weight gain. It was also clear that the determination of plasma amino acids, combined with other evaluation techniques in research and development, may serve as biomarkers for neuropsychiatric disorders, as well as preventive measures and the establishment of a new perspective for future targets adjuvant therapy for neuropsychiatric drugs.

Orientador: Márcio Alberto Torsoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:21:53Z (GMT). No. of bitstreams: 1 Pereira_ViniciusDias_M.pdf: 1179657 bytes, checksum: 3d71a3a82020acdf4b2e2a10b8eabfdf (MD5) Previous issue date: 2013 / Resumo: A AMPK hipotalâmica age como um sensor energético e é capaz de modular a ingestão alimentar, homeostase de glicose e a biossíntese de ácidos graxos. É conhecido que a injeção intra-hipotalâmica de ácidos graxos suprime a produção de glicose pelo fígado, principalmente pela ativação de canais de potássio sensíveis a ATP hipotalâmico (K(ATP)). Uma vez que em todos os modelos estudados a biossíntese de malonil-CoA estava envolvida, nós hipotetizamos que a Acetil-CoA Carboxilase poderia modular respostas contra-regulatórias independente da disponibilidade de nutrientes. Nesse estudo foram empregados os seguintes métodos: Immunoblot, PCR em tempo real, ELISA e avaliações bioquímicas. Através desses métodos, nós mostramos que a redução da expressão de acetil-CoA carboxilase pela injeção de oligonucleotídeo antisense intraventricular resultou no aumento da ingestão alimentar e diminuiu a expressão dos mRNA de CART, CRH e TRH. Além disso, como nos ratos em jejum, os ratos tratados com oligonucleotídeo antisense apresentaram concentrações de corpos cetônicos e glucagon séricos aumentados, além de níveis de insulina e glicogênio hepático diminuídos. A redução de acetil-CoA carboxilase hipotalâmica também aumentou a expressão de PEPCK, fosforilação de AMPK e a produção de glicose no fígado. Interessantemente, esses efeitos foram observados sem modificação da fosforilação da AMPK hipotalâmica. Com isso, concluímos que a inibição da ACC hipotalâmica pode ativar resposta contra-regulatória hepática independente da ativação da AMPK hipotalâmica / Abstract: BACKGROUND: Hypothalamic AMPK acts as a cell energy sensor and can modulate food intake, glucose homeostasis, and fatty acid biosynthesis. Intrahypothalamic fatty acid injection is known to suppress liver glucose production, mainly by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels. Since all models employed seem to involve malonyl-CoA biosynthesis, we hypothesized that acetyl-CoA carboxylase can modulate the counter-regulatory response independent of nutrient availability. METHODOLOGY/PRINCIPAL FINDINGS: In this study employing immunoblot, realtime PCR, ELISA, and biochemical measurements, we showed that reduction of the hypothalamic expression of acetyl-CoA carboxylase by antisense oligonucleotide after intraventricular injection increased food intake and diminished the expression of CART, CRH, and TRH mRNA. Additionally, as in fasted rats antisense oligonucleotide-treated rats, increased serum glucagon and ketone bodies were observed along with diminished levels of serum insulin and hepatic glycogen. The reduction of hypothalamic acetyl-CoA carboxylase also increased PEPCK expression, AMPK phosphorylation, and glucose production. Interestingly, these effects were observed without modification of hypothalamic AMPK phosphorylation. CONCLUSION/SIGNIFICANCE: Hypothalamic ACC inhibition can activate hepatic counter-regulatory response independent of hypothalamic AMPK activation / Mestrado / Clinica Medica / Mestre em Clinica Medica

Gonadotropin-releasing hormone (GnRH) and its superactive analogues are currently being used in the treatment of a number of endocrine disorders, such as endometriosis, precocious puberty, infertility and prostatic cancer. Selection of these analogues for clinical use have been previously based on their activities in animal models. This thesis has therefore investigated the binding characteristics of the human GnRH receptor, in comparison to those of the rat receptor, as well as the activities of a number of GnRH analogues for stimulating luteinising hormone (LH) and follicle stimulating hormone (FSH) secretion from cultured human pituitary cells. The establishment of a human pituitary bioassay system has further made possible the investigation of the direct regulatory roles of GnRH and other neuropeptides in man. To date, such studies in man have been performed in vivo and are thus complicated by the simultaneous interactions of numerous modulators.

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.:Background; Methods; Results; Conclusions

The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations. The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation. DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression.

Indiana University-Purdue University Indianapolis (IUPUI) / The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are peptides implicated in the inflammatory response. They are released in increased amounts during inflammation and induce thermal hyperalgesia. Whether these molecules directly affect the sensitivity of primary nociceptive sensory neurons is unknown. This information could provide a link between increased inflammation-induced release of GFLs and their ability to promote inflammatory hyperalgesia. These molecules bind to one of four GFRα receptor subtypes, and this GFL-GFRα complex often translocates to the receptor tyrosine kinase, Ret. The focus of this dissertation was to determine whether GFLs modulate the stimulated-release of calcitonin gene-related peptide (CGRP). Isolated sensory neurons and freshly dissociated spinal cord tissue were used to examine the enhancement in stimulated-release of CGRP, a measure of sensitization. Exposure of isolated sensory neurons to GDNF, neurturin, and artemin, enhanced the capsaicin stimulated-release of immunoreactive CGRP (iCGRP). Sensitization by GFLs occurred in freshly dissociated spinal cord tissue. Persephin, another member of the GFL family, did not enhance stimulated-release of iCGRP. These results demonstrate that specific GFLs are mediators of neuronal sensitivity. The intracellular signaling pathways responsible for this sensitization were also evaluated. Inhibition of the mitogen activated protein kinase (MAPK)/extracellular signal-related kinase 1/2 (Erk 1/2) pathway selectively abolished the enhancement of CGRP release by GDNF. NTN-induced sensitization was abolished by inhibition of the phosphatidylinositol-3-kinase (PI-3K) pathway. Reduction in Ret abolished the GDNF-induced sensitization, but did not fully inhibit NTN or ART-induced sensitization. Inhibition of other cell surface receptors (neural cell adhesion molecule (NCAM), and Integrin β-1) had distinct effects on the sensitization capability of each of the GFLs. Ret and NCAM inhibition in combination abolished ART-induced sensitization. It was necessary to inhibit Ret, NCAM, and Integrin β-1 to prevent the NTN-induced sensitization. These data demonstrate that the GFLs use distinct signaling mechanisms to induce the sensitization of nociceptive sensory neurons. The work presented in this thesis provides the first evidence for these novel and distinct Ret-independent pathways for GFL-induced actions and provides insight into the mechanism of sensory neuronal sensitization in general.

Indiana University-Purdue University Indianapolis (IUPUI) / Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.