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The objectives of this work were to investigate the nutritional and physicochemical characteristics as well as the functional properties of quinoa protein isolates (QPI) from different varieties, and to determine their potential use of such protein isolates in food products. Proteins were isolated by isoelectric precipitation at pH 5 from quinoa flour, and the QPI had a protein percentage of over 85%. The comparison of the flours and QPI electrophoretic profiles indicated that the extraction method allowed isolating practically all proteins of each variety. All the varieties analyzed had high lysine content, compared with cereals, and the essential amino acid content of Bolivian varieties was higher than varieties from Peru. The pH value affected the solubility and foaming capacity, and the magnitude of effects depended on the variety. Cluster analysis showed a strong influence of variety source and amino acid composition on protein physicochemical and functional properties; samples from Bolivia (cluster 2) were characterized as having the highest thermal stability, oil binding capacity, and water binding capacity at acid pH; samples from Peru (cluster 1) had the highest water binding capacity at basic pH and foaming capacity at pH 5. QPI presented a potential as an alternative vegetable protein for food application, in particular for vegetarian and vegan diets. [ABSTRACT FROM AUTHOR]

Six novel asymmetrical monomethine were synthesized via the of 1-butyl-2-(methylthio)benzo[ c, d]indol-1-ium iodide and various 1,5-substituted indolenine salts under basic conditions. The dyes were characterized using UV-vis spectroscopy, fluorescence, 1H NMR, 13C NMR, and ; furthermore, the purity of these compounds was observed using LC/ELSD/MS. [ABSTRACT FROM AUTHOR]

Although it was originally characterized as a constituent of focal adhesions in fibroblasts, focal adhesion kinase ( FAK) is now considered to be not only a mediator of adhesion processes but also a crucial regulator of guidance and a modulator of gene expression. FAK is the main transducer of the integrin signaling required to stabilize the actin cytoskeleton. However, additional activities have been described over the years. In the brain, FAK deserves particular attention as it is found in various alternatively spliced forms - these distributed in multiple subcellular compartments or bound to multiple partners. Moreover, its signaling involves not only phosphorylation but also ubiquitination and proteolysis. Several experimental cell models demonstrate that FAK increases or decreases migration, participates in differentiation and contributes to plasticity events. In addition, this kinase is linked to cell survival in cancer and apoptosis. This review focuses on the diversity of events involving brain-located forms of FAK. [ABSTRACT FROM AUTHOR]

The reelin signaling protein and its downstream components have been associated with synaptic plasticity and neurotransmission. The reelin signaling pathway begins with the binding of reelin to the transmembrane lipoprotein receptor apolipoprotein E receptor 2 (ApoER2), which in turns induces the sequential cleavage of ApoER2 by the sequential action of a- and ?-secretases. Using conditional-knockout mice of the catalytic component of the ?-secretase complex, presenilin 1 (PS1), we demonstrated increased brain ApoER2 and reelin protein and transcript levels, with no changes in the number of reelin-positive cells. Using the human SH-SY5Y neuroblastoma cell line, we showed that ApoER2 processing occurs in the presence of PS1, producing an intracellular ApoER2 C-terminal fragment. In addition, the pharmacologic inhibition of ?-secretase in SH-SY5Y cells led to increased reelin levels. Overexpression of ApoER2 decreased reelin mRNA levels in these cells. A luciferase reporter gene assay and nuclear fractionation confirmed that increased amounts of intracellular fragment of ApoER2 suppressed reelin expression at a transcriptional level. Chromatin immunoprecipitation experiments corroborated that the intracellular fragment of ApoER2 bound to the RELN promoter region. Our study suggests that PS1/?-secretase-dependent processing of the reelin receptor ApoER2 inhibits reelin expression and may regulate its signaling. [ABSTRACT FROM AUTHOR]

Patients with Alzheimer's disease (AD) display altered functioning of cortical networks, including altered patterns of synchronous activity and a serious deficit in cholinergic septohippocampal (SH) innervation. However, the mechanisms underlying these alterations and the implication of the GABAergic SH component in AD are largely unknown. In addition, the GABAergic septohippocampal pathway (SHP) is believed to regulate synchronous hippocampal activity by controlling the activity of interneurons. Here we show, using well-characterized pathway tracing experiments, that innervation of the GABAergic SHP decreases during normal aging. Furthermore, in an AD mouse model (hAPPSw,Ind; J20 mice), the GABAergic SHP shows a dramatic and early onset of this decrease in 8-mo-old mice. This decline is not caused by neuronal loss, but by the reduced number and complexity of GABAergic SH axon terminals. Finally, we demonstrate that hippocampal θ and γ rhythm power spectra are markedly diminished in 8-mo-old behaving mice expressing mutated hAPP. In addition to the well-known loss of cholinergic input to the hippocampus in AD, these data suggest that the altered patterns of synchronous activity seen in patients with AD could be caused by the loss of GABAergic SH axons, which modulate hippocampal network activities. [ABSTRACT FROM AUTHOR]

Directed cell migration and axonal guidance are essential steps in neural development that share many molecular mechanisms. The guidance of developing axons and migrating neurons is likely to depend on the precise control of plasmalemma turnover in selected regions of leading edges and growth cones, respectively. Previous results provided evidence of a signaling mechanism that couples chemotropic deleted in colorectal cancer (DCC)/Netrin-1 axonal guidance and exocytosis through Syntaxin1(Sytx1)/TI-VAMP SNARE proteins. Here we studied whether Netrin-1-dependent neuronal migration relies on a similar SNARE mechanism. We show that migrating neurons in the lower rhombic lip (LRL) express several SNARE proteins, and that DCC co-associates with Sytx1 and TI-VAMP in these cells. We also demonstrate that cleavage of Sytx1 by botulinum toxin C1 (BoNT/C1) abolishes Netrin-1-dependent chemoattraction of migrating neurons, and that interference of Sytx1 functions with shRNAs or Sytx1-dominant negatives disrupts Netrin-1-dependent chemoattraction of LRL neurons. These findings indicate that a Sytx1/DCC interaction is required for Netrin-1 guidance of migrating neurons, thereby highlighting a relationship between guidance signaling and SNARE proteins that regulate membrane turnover. [ABSTRACT FROM AUTHOR]

The septohippocampal (SH) pathway comprises cholinergic and GABAergic fibers. Whereas the former establish synaptic contacts with all types of hippocampal neurons, the latter form complex baskets specifically on interneurons. The GABAergic SH function is associated with the control of hippocampal synchronous networks. Little is known about the mechanisms involved in the formation of the GABAergic SH pathway. Semaphorin (Sema) 3C is expressed in most hippocampal interneurons targeted by these axons. To ascertain whether Sema 3C influences the formation of the SH pathway, we analyzed the development of this connection in Sema 3C-deficient mice. As these animals die at birth, we developed an in vitro organotypic co-culture model reproducing the postnatal development of the SH pathway. In these SH co-cultures, the GABAergic SH pathway developed with target specificity similar to that present in vivo. SH axons formed incipient baskets on several types of hippocampal interneurons at 7 days in vitro, which increased their complexity by 18-25 days in vitro. These SH fibers formed symmetric synaptic contacts on GABAergic interneurons. This synaptic specificity was not influenced by the absence of entorhinal afferents. Finally, the absence of Sema 3C in target neurons or its blockage by neuropilin-1 and -2 ectodomains in slice co-cultures did not lead to major changes in either the target specificity of the GABAergic SH pathway or its density of innervation. We conclude that the formation and synaptic specificity of the GABAergic SH pathway relies on robust molecular mechanisms, independent of Sema 3C, that are retained in our in vitro co-culture model. [ABSTRACT FROM AUTHOR]

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin-malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 113, 1644–1658. Lesioned axons do not regenerate in the adult mammalian CNS, owing to the over-expression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1–40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3β (GSK3β) and extracellular-related kinase (ERK) 1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3β and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: (i) cerebellar granule cells and (ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3β inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Finally, these regenerative effects were corroborated in the lesioned entorhino-hippocampal pathway in NgR1−/ − mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections. [ABSTRACT FROM AUTHOR]

Prnp knockout mice that overexpress an amino-truncated form of PrPc (ΔPrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl-2 overexpression in neurons on the development of the neurological syndrome and cerebellar pathology of ΔPrP. We show that ΔPrP overexpression activates the stress-associated kinases ERK1-2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of ΔPrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase-3 activation and cell death, which can be prevented by coexpressing Bcl-2. Finally, we corroborate in vivo that neuronal-directed Bcl-2 overexpression in ΔPrP mice (ΔPrP Bcl-2) markedly reduces caspase-3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy. [ABSTRACT FROM AUTHOR]

Abstract: Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106–126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106–126) in cortical neurons and the participation of PrPc. We show that PrP(106–126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects. [Copyright &y& Elsevier]

In examining the role of Class 3 secreted semaphorins in the prenatal and postnatal development of the septohippocampal pathway, we found that embryonic (E14-E16) septal axons were repelled by the cingulate cortex and the striatum. We also found that the hippocampus exerts chemorepulsion on dorsolateral septal fibers, but not on fibers arising in the medial septum/diagonal band complex, which is the source of septohippocampal axons. These data indicate that endogenous chemorepellents prevent the growth of septal axons in nonappropriate brain areas and direct septohippocampal fibers to the target hippocampus. The embryonic septum expressed np-1 and np-2 mRNAs, and the striatum and cerebral cortex expressed sema 3A and sema 3F. Experiments with recombinant semaphorins showed that Sema 3A and 3F, but not Sema 3C or 3E, induce chemorepulsion of septal axons. Sema 3A and 3F also induce growth cone collapse of septal axons. This indicates that these factors are endogenous cues for the early guidance of septohippocampal fibers, including cholinergic and γ-aminobutyric acid (GABA)ergic axons, during the embryonic stages. During postnatal stages, when target cell selection and synaptogenesis take place, np-1 and np-2 were expressed by septohippocampal neurons at all ages tested. In the target hippocampus, pyramidal and granule cells expressed sema 3E and sema 3A, whereas most interneurons expressed sema 3C, but few expressed sema 3E or 3A. Combined tracing and expression studies showed that GABAergic septohippocampal fibers terminated preferentially onto sema 3C-positive interneurons. In contrast, cholinergic septohippocampal fibers terminated onto sema 3E and sema 3A-expressing pyramidal and granule cells. The data suggest that Class 3 secreted semaphorins are involved in postnatal development. Moreover, because GABAergic and cholinergic axons terminate onto neurons expressing distinct, but overlapping, patterns of semaphorin expression, semaphorin functions may be regulated by different signaling mechanisms at postnatal stages. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine-preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle. [ABSTRACT FROM AUTHOR]

Abstract Synchronized activity of neuronal networks has been proposed to be essential for cerebellar function. To examine the occurrence and organization of spontaneous neuronal activity in the cerebellum in vivo , we imaged mouse cerebellar slices loaded with the intracellular Ca2+ concentration indicator, fura-2. Recordings were then analysed statistically to identify correlated network activity. Ca2+ imaging revealed consistent spontaneous correlated network activity of granule cells (GC), which often occurred in clusters of coactivated GC. The number of spontaneously active GC, their activation frequency and correlation, were controlled by glutamate and GABA ionotropic receptors. These findings indicate that distinctive patterns of correlated activity between GC networks may be relevant for cerebellar circuit function. Cannabinoid antagonist-precipitated Δ9 -tetrahydrocannabinol (THC) withdrawal impaired motor coordination. Given that the cerebellum has been suggested recently to be a main substrate for cannabinoid withdrawal, we used imaging of spontaneous network activity to examine whether GC, which contain CB1 cannabinoid receptors, respond to chronic THC treatment and withdrawal. Acute administration of THC had no effect on patterns of spontaneous GC network activity. In contrast, chronic THC administration severely impaired GC activity and network coordination. Incubation of cerebellar slices, from chronically THC-treated mice, with the cannabinoid antagonist, SR141716A increased the number and network correlation of active GC. These data provide physiological evidence of the involvement of cerebellar circuits in the adaptive changes occurring during chronic THC exposure and withdrawal. [ABSTRACT FROM AUTHOR]

Severed adult CNS axons can extend over long distances when a permissive 'milieu', such as grafted Schwann cells or ensheathing cells, is provided. Moreover, functional blocking of endogenous inhibitory factors, such as Nogo-A or proteoglycans, enhances the regeneration of axotomized neurons. Here we examine whether guidance cues available during the development of axonal pathways could also potentiate the regeneration of lesioned adult circuits. The CajaI-Retzius cells in the hippocampus are transient pioneer neurons that guide entorhino-hippocampal afferents to their target layers. By using an in vitro model of axotomy of the entorhino-hippocampal pathway we show that CajaI-Retzius cells triggered the regeneration of the axotomized entorhinohippocampal pathway. Furthermore, the regrowth induced by CajaI-Retzius cells was robust and its pattern was indistinguishable from that of the unlesioned entorhino-hippocampal pathway. Thus, regenerating axons regrew in a layer-specific fashion towards the appropriate target layers, making synaptic contacts with target pyramidal neurons. Interestingly, the ability of lesioned entorhinal axons to regrow was maintained for at least 9 days after axotomy. These results show that the growth-promoting cells controlling the development of neural circuits will be a relevant approach to promoting the regeneration of lesioned adult CNS pathways. [ABSTRACT FROM AUTHOR]

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Presents findings of a study on the regeneration of the entorhino-hippocampal (EHP) pathway in vitro through the combination of extrinsic strategies targeting cold-shock protein G (CSPG) and Nogo-A binding. Proliferation of glial cells after EHP axotomy in vitro; Overexpression of CSPG, Nogo-A and NgR; Effect of chondroitinase ABC (ChABC) and the peptide NEP-40 on axonal regeneration in vitro; Efficiency of a delayed ChABC and NEP-40 treatment after EHP lesion.