Altered hippocampal doublecortin expression in Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by motor and non-motor symptoms. Motor symptoms include bradykinesia, resting tremors, muscular rigidity, and postural instability, while non-motor symptoms include cognitive impairments, mood disturbances, sleep disturbances, autonomic dysfunction, and sensory abnormalities. Some of these symptoms may be influenced by the proper hippocampus functioning, including adult neurogenesis. Doublecortin (DCX) is a microtubule-associated protein that plays a pivotal role in the development and differentiation of migrating neurons. This study utilized postmortem human brain tissue of PD and age-matched control individuals to investigate DCX expression in the context of adult hippocampal neurogenesis. Our findings demonstrate a significant reduction in the number of DCX-expressing cells within the subgranular zone (SGZ), as well as a decrease in the nuclear area of these DCX-positive cells in postmortem brain tissue obtained from PD cases, suggesting an impairment in the adult hippocampal neurogenesis. Additionally, we found that the nuclear area of DCX-positive cells correlates with pH levels. In summary, we provide evidence supporting that the process of hippocampal adult neurogenesis is likely to be compromised in PD patients before cognitive dysfunction, shedding light on potential mechanisms contributing to the neuropsychiatric symptoms observed in affected individuals. Understanding these mechanisms may offer novel insights into the pathophysiology of PD and possible therapeutic avenues.

Ecdysis-related pleiotropic neuropeptides expression during Anopheles albimanus development.

OBJECTIVE: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. MATERIALS AND METHODS: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. RESULTS: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. CONCLUSIONS: The CCAP, corazonin and ETH neuropeptidesare potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Ecdysis-related pleiotropic neuropeptides expression during Anopheles albimanus development / Expresión de neuropéptidos pleiotrópicos asociados con ecdisis durante el desarrollo del mosquito Anopheles albimanus

Abstract: Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Resumen: Objetivo: Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus. Material y métodos: Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR. Resultados. A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito. Conclusión. Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.

Orexin and neuropeptide Y: tissue specific expression and immunoreactivity in the hypothalamus and preoptic area of the cichlid fish Cichlasoma dimerus.

Neuropeptide Y (NPY) and orexin are neuropeptides involved in the regulation of feeding in vertebrates. In this study we determined the NPY and orexin mRNA tissue expression and their immunoreactivity distribution in both preoptic area and hypothalamus, regions involved in the regulation of feeding behavior. Both peptides presented a wide expression in all tissues examined. The NPY-immunoreactive (ir) cells were localized in the ventral nucleus posterioris periventricularis (NPPv) and numerous ir-NPY fibers were found in the nucleus lateralis tuberis (NLT), the nucleus recess lateralis (NRL) and the neurohypophysis. Ir-orexin cells were observed in the NPPv, dorsal NLT, ventral NLT, lateral NLT (NLTl) and the lateral NRL. Ir-orexin fibers were widespread distributed along all the hypothalamus, especially in the NLTl. Additionally, we observed the presence of ir-orexin immunostaining in adenohypophyseal cells, especially in somatotroph cells and the presence of a few ir-orexin-A fibers in the neurohypophysis. In conclusion, both peptides have an ubiquitous mRNA tissue expression and are similarly distributed in the hypothalamus and preoptic area of Cichlasoma dimerus. The presence of ir-orexin in adenohypohyseal cells and the presence of ir-orexin and NPY fibers in the neurohypophysis suggest that both peptides may play an important neuroendocrine role in anterior pituitary.

The transcription factor chicken Scratch2 is expressed in a subset of early postmitotic neural progenitors.

Scratch proteins are members of the Snail superfamily which have been shown to regulate invertebrate neural development. However, in vertebrates, little is known about the function of Scratch or its relationship to other neural transcription factors. We report the cloning of chicken Scratch2 (cScrt2) and describe its expression pattern in the chick embryo from HH15 through HH29. cScrt2 was detected in cranial ganglia, the nasal placode and neural tube. At all stages examined, cScrt2 expression is only detected within a subregion of the intermediate zone of the neural tube. cScrt2 is also expressed in the developing dorsal root ganglia from HH22-23 onwards and becomes limited to its dorsal medial domain at HH29. phospho-Histone H3 and BrdU-labeling revealed that the cScrt2 expression domain is located immediately external to the proliferative region. In contrast, cScrt2 domain overlapped almost completely with that of the postmitotic neural transcription factor NeuroM/Ath3/NEUROD4. Together, these data define cScrt2-positive cells as a subset of immediately postmitotic neural progenitors. Previous data has shown that Scrt2 is a repressor of E-box-driven transcription whereas NeuroM is an E-box-transactivator. In light of these data, the co-localization detected here suggests that Scrt2 and NeuroM may have opposing roles during definition of neural subtypes.

Distribution and characterization of doublecortin-expressing cells and fibers in the brain of the adult pigeon (Columba livia).

Doublecortin (DCX) is a microtubule-associated protein essential for the migration of immature neurons in the developing and adult vertebrate brain. Herein, the distribution of DCX-immunoreactive (DCX-ir) cells in the prosencephalon of the adult pigeon (Columba livia) is described, in order to collect the evidence of their immature neural phenotype and to investigate their putative place of origin. Bipolar and multipolar DCX-ir cells were observed to be widespread throughout the parenchyma of the adult pigeon forebrain. Small, bipolar and fusiform DCX-ir cells were especially concentrated at the tips of the lateral walls of the lateral ventricles (VZ) and sparsely distributed in the remaining ependyma. Multipolar DCX-ir cells populated the pallial regions. None of these DCX-ir cells seemed to co-express NeuN or GFAP, suggesting that they were immature neurons. Two different migratory-like routes of DCX-ir cells from the VZ toward different targets in the parenchyma were putatively identified: (i) rostral migratory-like bundle; and (ii) lateral migratory-like bundle. In addition, pial surface bundles and intra-ependymal fascicles were also observed. Pigeons treated with 5-bromo-desoxyuridine (BrdU, 3 intraperitoneal injections of 100mg/kg 2h apart, sacrificed 2h after last injection) displayed BrdU-immunoreactive cells (BrdU-ir) in VZ and ependyma whereas the parenchyma was free of such cells. Despite the regional overlapping, there was no evidence of double-labeling between BrdU and DCX. Therefore, the VZ in the brain of adult pigeons seems to have rapidly dividing cells as putative progenitors of newborn neurons populating the forebrain. The distribution of the newborn neurons in the avian prosencephalon and their migration pathways appear to be larger than in mammals, suggesting that the morphological turnover of forebrain circuits is an important mechanism for brain plasticity in avian species during adulthood.

Differential effects of swimming training on neuronal calcium sensor-1 expression in rat hippocampus/cortex and in object recognition memory tasks.

Physical activity has been proposed as a behavioral intervention that improves learning and memory; nevertheless, the mechanisms underlying these health benefits are still not well understood. Neuronal Calcium Sensor-1 (NCS-1) is a member of a superfamily of proteins that respond to local Ca(2+) changes shown to have an important role in learning and memory. The aim of the present study was to investigate the effects of swimming training on NCS-1 levels in the rat brain after accessing cognitive performance. Wistar rats were randomly assigned to sedentary (SG) or exercised groups (EG). The EG was subject to forced swimming activity, 30 min/day, 5 days/week, during 8 weeks. Progressive load trials were performed in the first and last week in order to access the efficiency of the training. After the 8 week training protocol, memory performance was evaluated by the novel object preference and object location tasks. NCS-1 levels were measured in the cortex and hippocampus using immunoblotting. The EG performed statistically better for the spatial short-term memory (0.73 ± 0.01) when compared to the SG (0.63 ± 0.02; P<0.05). No statistically significant exercise-effect was observed in the novel object preference task (SG 0.65 ± 0.02 and EG 0.68 ± 0.02; p>0.05). In addition, chronic exercise promoted a significant increase in hippocampal NCS-1 levels (1.8 ± 0.1) when compared to SG (1.17 ± 0.08; P<0,05), but had no effect on cortical NCS-1 levels (SG 1.6 ± 0.1 and EG 1.5 ± 0.1; p>0.05). Results suggest that physical exercise would modulate the state of the neural network regarding its potential for plastic changes: physical exercise could be modulating NCS-1 in an activity dependent manner, for specific neural substrates, thus enhancing the cellular/neuronal capability for plastic changes in these areas; which, in turn, would differentially effect ORM task performance for object recognition and displacement.

Glucocorticoids are required for meal-induced changes in the expression of hypothalamic neuropeptides.

Glucocorticoid deficiency is associated with a decrease of food intake. Orexigenic peptides, neuropeptide Y (NPY) and agouti related protein (AgRP), and the anorexigenic peptide proopiomelanocortin (POMC), expressed in the arcuate nucleus of the hypothalamus (ARC), are regulated by meal-induced signals. Orexigenic neuropeptides, melanin-concentrating hormone (MCH) and orexin, expressed in the lateral hypothalamic area (LHA), also control food intake. Thus, the present study was designed to test the hypothesis that glucocorticoids are required for changes in the expression of hypothalamic neuropeptides induced by feeding. Male Wistar rats (230-280 g) were subjected to ADX or sham surgery. ADX animals received 0.9% NaCl in the drinking water, and half of them received corticosterone in the drinking water (B: 25 mg/L, ADX+B). Six days after surgery, animals were fasted for 16 h and they were decapitated before or 2 h after refeeding for brain tissue and blood collections. Adrenalectomy decreased NPY/AgRP and POMC expression in the ARC in fasted and refed animals, respectively. Refeeding decreased NPY/AgRP and increased POMC mRNA expression in the ARC of sham and ADX+B groups, with no effects in ADX animals. The expression of MCH and orexin mRNA expression in the LHA was increased in ADX and ADX+B groups in fasted condition, however there was no effect of refeeding on the expression of MCH and orexin in the LHA in the three experimental groups. Refeeding increased plasma leptin and insulin levels in sham and ADX+B animals, with no changes in leptin concentrations in ADX group, and insulin response to feeding was lower in this group. Taken together, these data demonstrated that circulating glucocorticoids are required for meal-induced changes in NPY, AgRP and POMC mRNA expression in the ARC. The lower leptin and insulin responses to feeding may contribute to the altered hypothalamic neuropeptide expression after adrenalectomy.

Guided motor training induces dendritic spine plastic changes in adult rat cerebellar purkinje cells.

The simple cerebellar lobule is involved in several neuromotor processes and it is activated during guided exercise. Although guided exercises are essential for motor rehabilitation, the plastic events that occur in the simple cerebellar lobule during motor training remain unknown. In this study, normal adult rats were intensely trained on a motorized treadmill during a period of four weeks (IT group) varying both the velocity and the slope of the moving belt, and they were compared to a mildly trained (MC) group and an intact control group (IC). Dendritic spine density and proportions of the different spine types on Purkinje cells was assessed in the cerebellar simple lobule, as was drebrin A expression. Both dendritic spine density and drebrin expression increased in the MC and IT groups. Stubby spines were more abundant in the MC animals, while there was an increase in both stubby and wide spines in IT rats. In addition, mushroom spines were more numerous in the IT group. Increases in stubby and wide spines could be related to regulation of the excitability in Purkinje cells due to the motor training regime experienced by the MC and IT rats. Moreover, the observed increase in mushroom spines in the IT group could be related with the motor adjustments imposed by training.

Neuropeptides in dental pulp: the silent protagonists.

Dental pulp is a soft mesenchymal tissue densely innervated by afferent (sensory) fibers, sympathetic fibers, and parasympathetic fibers. This complexity in pulp innervation has motivated numerous investigations regarding how these 3 major neuronal systems regulate pulp physiology and pathology. Most of this research is focused on neuropeptides and their role in regulating pulpal blood flow and the development of neurogenic inflammation. These neuropeptides include substance P, calcitonin gene-related peptide, neurokinin A, neuropeptide Y, and vasoactive intestinal polypeptide among others. The purpose of this article is to review recent advances in neuropeptide research on dental pulp, including their role in pulp physiology, their release in response to common dental procedures, and their plasticity in response to extensive pulp and dentin injuries. Special attention will be given to neuropeptide interactions with pulp and immune cells via receptors, including studies regarding receptor identification, characterization, mechanisms of action, and their effects in the development of neurogenic inflammation leading to pulp necrosis. Their role in the growth and expansion of periapical lesions will also be discussed. Because centrally released neuropeptides are involved in the development of dental pain, the pain mechanisms of the pulpodentin complex and the effectiveness of present and future pharmacologic therapies for the control of dental pain will be reviewed, including receptor antagonists currently under research. Finally, potential clinical therapies will be proposed, particularly aimed to manipulate neuropeptide expression or blocking their receptors, to modulate a variety of biologic mechanisms, which preliminary results have shown optimistic results.

Methylphenidate alters NCS-1 expression in rat brain.

Methylphenidate has been used as an effective treatment for attention deficit hyperactivity disorder (ADHD). Methylphenidate (MPH) blocks dopamine and norepinephrine transporters causing an increase in extracellular levels. The use of psychomotor stimulants continues to rise due to both the treatment of ADHD and illicit abuse. Methylphenidate sensitization mechanism has still poor knowledge. Neuronal calcium sensor 1 was identified as a dopaminergic receptor interacting protein. When expressed in mammalian cells, neuronal calcium sensor 1 attenuates dopamine-induced D2 receptor internalization by a mechanism that involves a reduction in D2 receptor phosphorylation. Neuronal calcium sensor 1 appears to play a pivotal role in regulating D2 receptor function, it will be important to determine if there are alterations in neuronal calcium sensor 1 in neuropathologies associated with deregulation in dopaminergic signaling. Then, we investigated if methylphenidate could alter neuronal calcium sensor 1 expression in five brain regions (striatum, hippocampus, prefrontal cortex, cortex and cerebellum) in young and adult rats. These regions were chosen because some are located in brain circuits related with attention deficit hyperactivity disorder. Our results showed changes in neuronal calcium sensor 1 expression in hippocampus, prefrontal cortex and cerebellum mainly in adult rats. The demonstration that methylphenidate induces changes in neuronal calcium sensor 1 levels in rat brain may help to understand sensitization mechanisms as well as methylphenidate therapeutic effects to improve attention deficit hyperactivity disorder symptoms.

DARPP-32 and NCS-1 expression is not altered in brains of rats treated with typical or atypical antipsychotics.

Dopamine-mediated neurotransmission imbalances are associated with several psychiatry illnesses, such as schizophrenia. Recently it was demonstrated that two proteins involved in dopamine signaling are altered in prefrontal cortex (PFC) of schizophrenic patients. DARPP-32 is a key downstream effector of intracellular signaling pathway and is downregulated in PFC of schizophrenic subjects. NCS-1 is a neuronal calcium sensor that can inhibit dopamine receptor D2 internalization and is upregulated in PFC of schizophrenic subjects. It is well known that dopamine D2 receptor is the main target of antipsychotic. Therefore, our purpose was to study if chronic treatment with typical or atypical antipsychotics induced alterations in DARPP-32 and NCS-1 expression in five brain regions: prefrontal cortex, hippocampus, striatum, cortex and cerebellum. We did not find any changes in DARPP-32 and NCS-1 protein expression in any brain region investigated.

NCS-1 expression in rat brain after electroconvulsive stimulation.

Although electroconvulsive therapy (ECT) has been used as a treatment for mental disorder since 1930s, little progress has been made towards understanding the mechanisms underlying its therapeutic and adverse effects. The aim of this work was to analyze the expression of NCS-1 (neuronal calcium sensor 1, a protein that was found to be altered in post-mortem prefrontal cortex of schizophrenic patients) in striatum, cortex, hippocampus and cerebellum of Wistar rats after acute or chronic electroconvulsive stimulation (ECS). Rats were submitted to a single stimulation (acute) or to a series of eight stimulations, applied one every 48 h (chronic). Animals were killed for collection of tissue samples at time zero, 30 min, 3, 12, 24 and 48 h after stimulation in the acute model and at the same time intervals after the last stimulation in the chronic model. Our results indicated that chronic ECS increased the expression of NCS-1 only in cerebellum. Such results on the expression of proteins involved in signaling pathways that are relevant for neuropsychiatric disorders and treatment, in particular ECT, can contribute to shed light on the mechanisms related to therapeutic and adverse effects.

Functional expression and purification of recombinant Tx1, a sodium channel blocker neurotoxin from the venom of the Brazilian "armed" spider, Phoneutria nigriventer.

Tx1 from the venom of the Brazilian spider, Phoneutria nigriventer, is a lethal neurotoxic polypeptide of M(r) 8600 Da with 14 cysteine residues. It is a novel sodium channel blocker which reversibly inhibits sodium currents in CHO cells expressing recombinant sodium (Nav1.2) channels. We cloned and expressed the Tx1 toxin as a thioredoxin fusion product in the cytoplasm of Escherichia coli. After semipurification by immobilized Ni-ion affinity chromatography, the recombinant Tx1 was purified by reverse phase chromatography and characterized. It displayed similar biochemical and pharmacological properties to the native toxin, and it should be useful for further investigation of structure-function relationship of Na channels.

Expression of a functional recombinant Phoneutria nigriventer toxin active on K+ channels.

PnTx3-1 is a peptide isolated from the venom of the spider Phoneutria nigriventer that specifically inhibits A-type K(+) currents (I(A)) in GH(3) cells. Here we used a bacterial expression system to produce an NH(2)-extended mutant of PnTx3-1 (ISEF-PnTx3-1) and tested whether the toxin is functional. The recombinant toxin was purified from bacterial extracts by a combination of affinity and ion-exchange chromatography. The recombinant toxin blocked A-type K(+) currents in GH(3) cells in a fashion similar to that observed with the wild-type toxin purified from the spider venom. These results suggest that recombinant cDNA methods provide a novel source for the production of functional Phoneutria toxins. The recombinant ISEF-PnTx3-1 should be useful for further understanding of the role of A-type K(+) currents in biological processes.

The role of the hypothalamic neuropeptides hypocretin/orexin in the sleep-wake cycle.

The novel neuropeptides hypocretin/orexin have recently been located on the lateral hypothalamus cells. This system has been linked to the regulation of both feeding and sleep, and recent studies have found an association between a defect in these neuropeptides and narcolepsy. We conducted a MEDLINE review of all the articles published since the discovery of hypocretin/orexin peptides, narrowing the field to the relationship between these neuropeptides and sleep. The finding of a deletion in the transcription of the hypocretin receptor 2 gene in narcoleptic Doberman pinschers and the development of a knockout of the hypocretin gene in mice pointed to the relevance of this system in the sleep-wake cycle. We provide further evidence of the role of the hypocretin/orexin system in narcolepsy and in sleep regulation and present an integrative model of the pathophysiology of narcolepsy. The discovery of the link between these peptides and narcolepsy opens new avenues to both the understanding of sleep mechanisms and therapeutic implications for sleep disorders.

Neuropeptídios e dermatologia / Neuropeptides and dermatology

Os autores apresentam novos conceitos científicos dosneuropeptídeos, que atuam como neurotransmissores, neuro moduladores e neuro hormônios. Sua participaçäo na imunidade cutânea, nos processos de cicatrizaçäo e doenças crônicas da pele, säo evidentes. Os neuropeptídeos agem como mensageiros químicos, interligando o cérebro com os receptores da pele. Existem mais de cinqüenta neuropeptídeos envolvidos na transmissäo de sinais entre as células nervosas e o sistema auto-imune. Säo produzidos na pele neuropeptídeos opióides, que atuariam nas dores e emoçöes, promovendo a integraçäo do sistema neuroimunocutâneo. Uma das propostas do trabalho é uma visäo integrativa da Dermatologia na medicina geral.

Sequence of the cDNA coding for the lethal neurotoxin Tx1 from the Brazilian "armed" spider Phoneutria nigriventer predicts the synthesis and processing of a preprotoxin.

A cDNA library was constructed from the venom glands of the Brazilian "armed" spider, Phoneutria nigriventer, and a clone coding for Tx1, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Tx1 is initially synthesized as a preprotoxin. Four segments (comprising the signal sequence, a short, 15-amino acid, glutamate-rich sequence, the functional toxin, and 2 glycine residues) can be distinguished. The structure of the preprotoxin and the proposed processing steps required to form the mature Tx1 toxin show similarities with the synthesis and processing of omega-agatoxin IA.