A wide-ranging Pseudomonas aeruginosa PeptideAtlas build: A useful proteomic resource for a versatile pathogen.

Pseudomonas aeruginosa is an important opportunistic human pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under different conditions and from diverse cellular compartments have been joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1, the highest coverage among bacterial PeptideAtlas datasets. The proteins included cover 89% of metabolic proteins, 72% of proteins involved in genetic information processing, 83% of proteins responsible for environmental information processing, more than 88% of the ones related to quorum sensing and biofilm formation, and 89% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. The manuscript describes the building of the PeptideAtlas and the contribution of the different proteomic data used. SIGNIFICANCE: Pseudomonas aeruginosa is among the most versatile human bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

PH domain leucine-rich repeat protein phosphatase 1 contributes to maintain the activation of the PI3K/Akt pro-survival pathway in Huntington's disease striatum.

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntington's disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (Hdh(Q111/Q111) mouse striatum and STHdh(Q111/Q111) cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF + or - , R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Bax deficiency promotes an up-regulation of Bim(EL) and Bak during striatal and cortical postnatal development, and after excitotoxic injury.

In this study we analyzed whether other members of the Bcl-2 family are regulated in the absence of Bax during the postnatal development of the striatum and cortex and after striatal excitotoxic lesion. Compared with wild-type animals, Bax knockout mice showed region- and time-dependent increases in pro-apoptotic proteins Bak and Bim(EL). Excitotoxicity induced in the adult striatum increased Bim(EL) in both genotypes whereas Bak and Bcl-x(L) were only increased in Bax knockout mice. However, translocation of Bim(EL) protein to the mitochondrial fraction, cytochrome c release and caspase-3 activation were only observed in wild-type striata. Furthermore, analysis of Bim null mutant mice showed that this protein is not essential to excitotoxicity-induced striatal cell death. In conclusion, our results show that in Bax deficient mice Bim(EL) and Bak are specifically regulated during postnatal development, suggesting that these proteins may participate in the compensatory mechanisms triggered in the absence of Bax. In contrast, Bax is required to induce apoptosis after excitotoxicity in the adult striatum.

Mice heterozygous for neurotrophin-3 display enhanced vulnerability to excitotoxicity in the striatum through increased expression of N-methyl-D-aspartate receptors.

The striatum is one of the brain areas most vulnerable to excitotoxicity, a lesion that can be prevented by neurotrophins. In the present study, intrastriatal injection of the N-methyl-d-aspartate receptor (NMDAR) agonist quinolinate (QUIN) was performed in mice heterozygous for neurotrophin-3 (NT3 +/-) or brain-derived neurotrophic factor (BDNF +/-) to analyze the role of endogenous neurotrophins on the regulation of striatal neurons susceptibility to excitotoxic injury. QUIN injection induced a decrease in dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) protein levels that was higher in NT-3 +/- than in BDNF+/- or wild type animals. This enhanced susceptibility was specific for enkephalin- and tachykinin-positive projection neurons, and also for parvalbumin-positive interneurons. However the excitotoxic damage in large interneurons was not modified in NT-3 +/- mice compared with wild type animals. This effect can be related to the regulation of NMDARs by endogenous NT-3. Thus, our results show that there is an age-dependent regulation of NMDAR subunits NR1 and NR2A, but not NR2B, in NT-3 +/- mice. The deficit of endogenous NT-3 induced a decrease in NR1 and NR2A subunits at postnatal day (P) 0 and P3 mice respectively, whereas an upregulation was observed in 12 week old NT-3 +/- mice. This differential effect was also observed after administration of exogenous NT-3. In primary striatal cultures, NT-3 treatment induced an enhancement in NR2A, but not NR2B, protein levels. However, intrastriatal grafting of NT-3 secreting-cells in adult wild type mice produced a down-regulation of NR2A subunit. In conclusion, NT-3 regulates the expression of NMDAR subunits modifying striatal neuronal properties that confers the differential vulnerability to excitotoxicity in projection neurons and interneurons in the striatum.

Cellular and molecular mechanisms involved in the selective vulnerability of striatal projection neurons in Huntington's disease.

Neurodegenerative disorders affecting the central nervous system, such as Alzheimer's disease, Parkinson's disease, Huntington's chorea (HD) and amyotrophic lateral sclerosis are characterized by the loss of selected neuronal populations. Another striking feature shared by these diseases is the deposition of proteinaceous inclusion bodies in the brain, which may be intracytoplasmatic or intranuclear, or even extracellular. However, the density and prevalence of aggregates are not always directly related to neurodegeneration. Although some of these diseases are the result of mutations in known proteins, with HD a clear example, the expression and location of the affected protein do not explain the selective neurodegeneration. Therefore, other intrinsic mechanisms, characteristic of each neuronal population, might be involved in the neurodegenerative process. In this review we focus on several proposed mechanisms such as excitotoxicity, mitochondrial dysfunction and altered expression of trophic factors, which could account for the pathogenesis of HD.

Neuroprotection by neurotrophins and GDNF family members in the excitotoxic model of Huntington's disease.

Huntington's disease is a neurodegenerative disorder characterized by a selective degeneration of striatal projection neurons, which deal with choreic movements. Neuroprotective therapy could be achieved with the knowledge of the specific trophic requirements of these neuronal populations. Thus, the induction of endogenous trophic response or the exogenous administration of neurotrophic factors may help to prevent or stop the progression of the illness. Excitotoxicity has been implicated in the etiology of Huntington's disease, because intrastriatal injection of glutamate receptor agonists reproduces some of the neuropathological features of this disorder. Activation of glutamate receptors in the striatum differentially regulates the expression of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), neurturin, and their receptors in the striatum and in its connections, cortex, and substantia nigra, showing a selective trophic response against excitotoxic insults. Transplantation of cells genetically engineered to release neurotrophic factors in the striatum has been used to study the neuroprotective effects of neurotrophin and GDNF family members in the excitotoxic model of Huntington's disease. Neurotrophins (brain-derived neurotrophic factor [BDNF], neurotrophin-3, and neurotrophin-4) protected striatal projection neurons against quinolinic or kainic acid treatment. However, GDNF family members showed a more specific action. Neurturin only protected gamma-aminobutyric acid (GABA)/enkephalinergic neurons that project to the external segment of the globus pallidus, whereas GDNF exerts its effects on GABA/substance P positive neurons, which project to the substantia nigra pars compacta and the internal segment of the globus pallidus. In conclusion, the trophic requirements of each population of striatal projection neurons are due to a complex interaction between several neurotrophic factors, such as neurotrophins and GDNF family members, which can be modified, in different pathological conditions. Moreover, these neurotrophic factors may be able to provide selective protection for basal ganglia circuits, which are affected in striatonigral degenerative disorders, such as Huntington's disease or multisystem atrophy.

Brain-derived neurotrophic factor (BDNF) mediates bone morphogenetic protein-2 (BMP-2) effects on cultured striatal neurones.

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily that have multiple functions in the developing nervous system. One of them, bone morphogenetic protein-2 (BMP-2), promotes the differentiation of cultured striatal neurones, enhancing dendrite growth and calbindin-positive phenotype. Bone morphogenetic proteins have been implicated in cooperative interactions with other neurotrophic factors. Here we examined whether the effects of BMP-2 on cultured striatal neurones are mediated or enhanced by other neurotrophic factors. BMP-2 had a cooperative effect with low doses of brain-derived neurotrophic factor or neurotrophin-3 (but not with other neurotrophic factors such as glial cell line-derived neurotrophic factor, neurturin or transforming growth factor-beta 2) on the number of calbindin-positive striatal neurones. Moreover, BMP-2 induced phosphorylated Trk immunoreactivity in cultured striatal neurones, suggesting that neurotrophins are involved in BMP-2 neurotrophic effects. The addition of TrkB-IgG or antibodies against brain-derived neurotrophic factor abolished the effects of BMP-2 on the number and degree of differentiation of calbindin-positive striatal neurones. Indeed, BMP-2 treatment increased brain-derived neurotrophic factor protein levels in treated cultures media and BDNF immunocytochemistry revealed that this neurotrophin was produced by neuronal cells. Taken together, these results indicate that brain-derived neurotrophic factor mediates the effects of BMP-2 on striatal neurones.

Neuroprotection of striatal neurons against kainate excitotoxicity by neurotrophins and GDNF family members.

Neurotrophic factors are regarded as potential therapeutic tools in neurodegenerative disorders. Here, we analysed the protective effects of brain-derived neurotrophic factor, neurotrophin-3, glial cell line-derived neurotrophic factor and neurturin against the excitotoxic damage induced by kainate in striatal neurons in vitro and in vivo. Our results show that the decrease in the number of cultured striatal calbindin-positive neurons induced by kainate was prevented by treatment with any of these factors. To characterize their protective effects in vivo, cell lines overexpressing brain-derived neurotrophic factor, neurotrophin-3, glial cell line-derived neurotrophic factor or neurturin were grafted into the striatum. We found that the numbers of striatal projection neurons (calbindin-positive) and striatal interneurons (parvalbumin- or choline acetyltransferase-positive) were differentially decreased after kainate lesion. These neurotrophic factors prevented the loss of striatal projection neurons and interneurons with differing efficiency: brain-derived neurotrophic factor was the most efficient, whereas neurturin was the least. Our findings show that brain-derived neurotrophic factor, neurotrophin-3, glial cell line-derived neurotrophic factor and neurturin have specific neuroprotective profiles in striatal neurons and indicate that they are specific modulators of the survival of distinct subsets of striatal neurons in pathophysiological conditions.

Expression of brain-derived neurotrophic factor in cortical neurons is regulated by striatal target area.

Changes in BDNF expression after different types of brain insults are related to neuroprotection, stimulation of sprouting, and synaptic reorganization. In the cerebral cortex, an autocrine-paracrine mechanism for BDNF has been proposed because the distribution patterns of BDNF and TrkB expression are almost identical. Moreover, cortical BDNF is anterogradely transported to the striatum, suggesting a role of BDNF in the functional interaction between the two brain regions. Here we have examined the expression of this neurotrophin in the cerebral cortex after various striatal lesions. Intrastriatal injection of quinolinate, kainate, 3-nitropropionic acid, or colchicine increased BDNF mRNA levels in cerebral cortex. In contrast, stimulation of neuronal activity in the striatum did not change cortical BDNF expression. Both excitatory amino acids increased BDNF expression in neurons of cortical layers II/III, V, and VI that project to the striatum. Moreover, grafting a BDNF-secreting cell line prevented both the loss of striatal neurons and the cortical upregulation of BDNF induced by excitotoxins. Because retrograde transport in the corticostriatal pathway was intact after striatal lesions, our results suggest that striatal damage upregulates endogenous BDNF in corticostriatal neurons by a transneuronal mechanism, which may constitute a protective mechanism for striatal and/or cortical cells.

Neurturin protects striatal projection neurons but not interneurons in a rat model of Huntington's disease.

Glial cell line-derived neurotrophic factor and neurturin are neurotrophic factors expressed in the striatum during development and in the adult rat. Both molecules act as target-derived neurotrophic factors for nigrostriatal dopaminergic neurons. While glial cell line-derived neurotrophic factor has also been described to have local trophic effects on striatal neurons, the effects of neurturin in the striatum have not yet been described. Here we examine whether neurturin protects striatal projection neurons (calbindin-positive) and interneurons (parvalbumin- or choline acetyltransferase-positive) in an animal model of Huntington's disease. A fibroblast cell line engineered to over-express neurturin was grafted into adult rat striatum 24h before quinolinate injection. In animals grafted with a control cell line, intrastriatal quinolinate injection reduced the number of calbindin-, parvalbumin- and choline acetyltransferase-positive neurons, seven days post-lesion. Intrastriatal grafting of neurturin-secreting cells protected striatal projection neurons, but not interneurons, from quinolinate excitotoxicity. This effect was much more robust than that reported previously for a glial cell line-derived neurotrophic factor-secreting cell line on striatal calbindin-positive neurons. However, intrastriatal grafting of glial cell line-derived neurotrophic factor- but not neurturin-secreting cells prevented the decrease in choline acetyltransferase activity induced by quinolinate injection. Taken together, our results show that neurturin- and glial cell line-derived neurotrophic factor-secreting cell lines have clearly differential effects on striatal neurons. Grafting of the neurturin-secreting cell line showed a more specific and efficient trophic effect on striatal projection neurons, the neuronal population most affected in Huntington's disease. Therefore, our results suggest that neurturin is a good candidate for the treatment of this neurodegenerative disorder.

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 prevent the death of striatal projection neurons in a rodent model of Huntington's disease.

Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntington's disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF-, NT-3-, or NT-415-secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF-secreting cell line pre-vented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67-, preproenkephalin-, and preprotachykinin A- but not prodynorphin-expressing neurons were protected by grafting of NT-3- or NT-4/5-secreting cells but with less efficiency than the BDNF-secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntington's disease.

Brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 differentially regulate the phenotype and prevent degenerative changes in striatal projection neurons after excitotoxicity in vivo.

To determine whether growth factors of the neurotrophin family are able to regulate the phenotype of striatal projection neurons, cell lines overexpressing brain-derived neurotrophic factor, neurotrophin-3 or neurotrophin-4/5 were intrastriatally grafted. Striatal projection neurons were examined for the regulation of their soma areas and for the expression of glutamate decarboxylase 67, preprotachykinin A, preproenkephalin and prodynorphin messenger RNAs by in situ hybridization. Brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 differentially regulated the soma area of projection neurons at different distances from the graft, but did not modify their messenger RNA levels. Neurotrophin-3 induced an increase in the soma area of preproenkephalin- and preprotachykinin A-positive neurons, brain-derived neurotrophic factor increased the soma area of only preprotachykinin A-positive neurons, while neurotrophin-4/5 did not produce any effect. Because atrophy and neuronal loss are hallmarks of Huntington's disease, we next examined whether neurotrophins prevent degenerative changes in a quinolinate model of Huntington's disease. Seven days after intrastriatal quinolinate injection, we observed a halo of cell loss around the injection sites, reduced soma area of glutamate decarboxylase 67-, preproenkephalin- and preprotachykinin A-positive neurons bordering the lesion, and a decrease in the messenger RNA levels of glutamate decarboxylase 67 and these neuropeptides. Grafting of cell lines expressing brain-derived neurotrophic factor, neurotrophin-3 or neurotrophin-4/5 reduced the size of the lesion for preproenkephalin-, preprotachykinin- and glutamate decarboxylase 67-, but not for prodynorphin-positive neurons. Moreover, the three neurotrophins prevented the atrophy of all projection neurons, and the lesion-induced decrease in preproenkephalin and preprotachykinin A messenger RNA levels. We conclude that neurotrophins differentially regulate the phenotype of striatal projection neurons and prevent degenerative changes. The higher efficiency of neurotrophin-3 suggests a potential therapeutic application of this molecule in neurological disorders affecting striatal projection neurons, such as Huntington's disease.

The neurotrophin receptors trkA, trkB and trkC are differentially regulated after excitotoxic lesion in rat striatum.

In the present work, we examined the time-dependent changes in trkA, trkB and trkC mRNA levels induced by the injection of glutamate receptor agonists into the striatum. Changes in trk mRNAs induced by quinolinate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate or 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were analyzed by a ribonuclease protection assay. All high-affinity neurotrophin receptors showed differential regulation after intrastriatal injury. Up-regulation of trkA expression was observed in kainate- or ACPD-injected striata at 10 and 24 h, respectively, whereas quinolinate injection induced down-regulation between 4 and 6 h after injury. Interestingly, all the excitatory amino acid receptor agonists induced up-regulation of trkB-kinase mRNA levels. This increase was maximal between 2 and 4 h after injection except in kainate injected striata, which showed the peak of expression at 10 h. In contrast, no changes in trkC mRNA expression were observed after striatal excitotoxic injury. In conclusion, our results show that trk receptor mRNA levels are differentially regulated by excitatory amino acid receptor agonists in the striatum, suggesting that changes in the levels of neurotrophin receptors might be involved either in synaptic plasticity processes or in neuronal protection in the striatal excitotoxic paradigm.

Intrastriatal grafting of a GDNF-producing cell line protects striatonigral neurons from quinolinic acid excitotoxicity in vivo.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor with a therapeutic potential in neurodegenerative disorders. GDNF is expressed in the adult striatum, but its signalling tyrosine kinase receptor, c-ret, has not been detected in this structure by in situ hybridization. In the present work, we first examined c-ret and GDNF receptor alpha 1 (GFR-alpha 1) expression using an RNAse protection assay, and found that both receptors are expressed in the adult rat striatum. We then examined whether GDNF was able to regulate the phenotype and/or prevent the degeneration of striatal projection neurons in a well-characterized model of excitotoxic damage. A fibroblast cell line, engineered to overexpress GDNF, was grafted in adult rats striatum 24 h before quinolinic acid (QUIN) injection. QUIN injection alone or in combination with the control cell line induced a loss of glutamic acid decarboxylase 67 (GAD)-, preprotachykinin A (PPTA)-, prodynorphin (DYN)- and preproenkephalin (PPE)-positive neurons. GDNF selectively prevented: (i) the loss of a subpopulation of striatonigral neurons expressing GAD and PPTA; (ii) the atrophy of PPTA-positive neurons; and (iii) the decrease in GAD, PPTA and DYN mRNA expression, after QUIN injection. Moreover, in unlesioned animals, GDNF increased the size of PPTA-positive neurons and up-regulated their mRNA levels. In contrast, GDNF showed no effect in intact or lesioned striatopallidal PPE-positive neurons. Thus, our findings show that GDNF selectively regulates the phenotype and protects striatonigral neurons from QUIN-induced excitotoxicity, suggesting that GDNF may be used for the treatment of striatonigral degenerative disorders, e.g. Huntington's disease and multiple system atrophy.

Differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 after excitotoxicity in a rat model of Huntington's disease.

In the present study we have evaluated changes in nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3 (NT-3) mRNA expression induced by different glutamate receptor agonists injected into the neostriatum. Up-regulation of NGF expression was observed at 24 h after intrastriatal quinolinate injection, an N-methyl-D-aspartate receptor agonist, and this increase was maintained up to 7 days after lesion. NGF up-regulation was also apparent in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) treatment from 6 to 16 h postinjection. Instead, BDNF was up-regulated only at 6 h after kainate or AMPA excitotoxicity. Interestingly, NT-3 mRNA was down-regulated from 10 to 16 h following AMPA lesion, while 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid injection enhanced NT-3 mRNA levels at 10 h. Our results show a specific neurotrophin response induced by stimulation of each glutamate receptor. These activity-dependent changes might be involved in neuronal plasticity processes and may underlie the differential vulnerability of striatal neurons observed in neurodegenerative disorders.

A brain-derived neurotrophic factor (BDNF) related system is involved in the maintenance of the polyinnervate Torpedo electric organ.

Target-derived molecules are essential for the maintenance of neuron survival. In the present work, we introduce the electric organ of Torpedo marmorata as a tool for the study of trophic interactions in a polyinervate system. This electric organ maintains a large number of cholinergic terminals on the postsynaptic cell surface. We have observed that a soluble extract derived from the electric organ induces the maturation of Xenopus oocytes injected with presynaptic plasma membranes (PSPM), indicating that a trophic system may exist. Moreover, we have detected a p75NGFR related protein in PSPM by Western blot analysis. These results suggest the presence of a neurotrophin-related system maintaining the polyinnervate electric organ. Furthermore, molecular experiments showed that the brain-derived neurotrophic factor (BDNF) is the neurotrophin operating in our model. Using degenerate oligonucleotides which comprise a conserved fragment of all neurotrophins, we have only amplified by polymerase chain reaction a BDNF fragment. In a similar way, we have amplified and cloned a fragment of the TrkB/C high affinity BDNF receptor. The fact that degenerate oligonucleotides only amplify BDNF allows us to conclude that the polyinnervation is maintained by this neurotrophin either alone or in combination with other trophic factors.

Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures.

The neuroprotective effect of tachykinins against excitotoxic death of cholinergic neurons was studied in rat striatal cell cultures. Quinolinic acid (QUIN) and kainic acid (KA) produced a dose dependent decrease in choline acetyltransferase activity, but KA was more potent. Our results show that substance P (SP) totally reversed the toxicity induced by 125 microM QUIN but not by 40 microM KA. This effect was also observed using protease inhibitors or a SP-analog resistant to degradation, [Sar9]-Substance P. The survival of neuron specific enolase- and acetylcholinesterase (AChE)-positive cells after treatment with QUIN alone or in the presence of SP was also examined. We observed that, while a decrease in total cell number produced by QUIN was not prevented by SP treatment, AChE-positive cells were rescued from the toxic damage. To characterize the SP protective effect we used more selective agonists of the three classes of neurokinin (NK) receptors. [Sar9, Met(O2)11]-Substance P (NK1 receptor agonist), [Nle10]-Neurokinin A (NK2 receptor agonist) or [Me-Phe7]-Neurokinin B (NK3 receptor agonist) were all able to block the toxic effect of QUIN on cholinergic activity. These results show that tachykinins provide an important protective support for striatal neurons, suggesting a possible therapeutical benefit in neurodegenerative disorders affecting cholinergic neurons.

Glial cell line-derived neurotrophic factor protects striatal calbindin-immunoreactive neurons from excitotoxic damage.

The neostriatum is one of the areas with relatively high levels of glial cell line-derived neurotrophic factor (GDNF) messenger RNA expression in the developing and adult brain. GDNF expression in the neostriatum has been suggested to be involved in promoting the survival of nigral dopaminergic neurons, acting as a target-derived neurotrophic factor. However, GDNF messenger RNA expression in the striatum starts several days before dopaminergic and other afferent neurons reach the striatum, suggesting additional trophic effects of this factor on striatal neurons. In the present report, we have examined whether GDNF is able to prevent the degeneration of striatal calbindin- and parvalbumin-immunoreactive neurons in a lesion model of Huntington's disease. Fischer 344 rat 3T3 fibroblast cell line expressing high levels of GDNF (F3A-GDNF) was used to assess the protective effect of this factor, on striatal neurons, against excitotoxicity. Quinolinate (34 nmol) was injected at two different coordinates, and calbindin, parvalbumin and tyrosine hydroxylase immunoreactivity were examined seven days after lesion. Dopaminergic afferents were spared after quinolinate injection, but the number of calbindin- and parvalbumin-immunoreactive neurons was decreased. Interestingly, implantation of F3A-GDNF cells increased the density of tyrosine hydroxylase staining in the intact and also in the quinolinate-lesioned striatum. Furthermore, GDNF partially protected calbindin- but not parvalbumin-immunoreactive neurons from quinolinate excitotoxicity. Instead, mock-transfected fibroblasts did not affect any of these parameters. Our results show that GDNF specifically protects a subpopulation of striatal calbindin-immunoreactive neurons against quinolinate lesion, suggesting that GDNF administration may have a potential therapeutic application in the prevention and treatment of striatonigral degenerative disorders.

Unilateral neonatal hippocampal lesion alters septal innervation and trophism of the entorhinal cortex.

It is generally assumed that central nervous system injury sustained during development produces less severe behavioral deficits than damage in the adult, due to increased plasticity of the immature brain. However, developmental plasticity may also exacerbate deficits, presumably through formation of anomalous connections. Previous studies showed that after unilateral neonatal, but not adult, electrolytic hippocampal lesion spatial memory is severely impaired. To determine whether the memory deficit is correlated with anatomical changes in a major hippocampal afferent system, the septal input, the anterograde tracer Phaeseolus vulgaris leucoagglutinin was injected into the medial septum 2 months after unilateral neonatal hippocampal lesion. The density of septal fiber projections into the entorhinal cortex (EC) was found to be increased. Choline-acetyltransferase activity increased significantly in the EC 2 months postlesion, suggesting that septal cholinergic fibers are sprouting. Finally, nerve growth factor (NGF), which can mediate sprouting, was measured in the EC, NGF protein increased transiently 7 to 12 days postlesion in the ipsilateral EC, suggesting that increased trophic support is associated with growth of septal afferents into the EC. Thus, neonatal hippocampal lesion causes a reorganization of axonal connections associated with elevated NGF in the target region of the increased septal input. Moreover, since previous studies showed that the neonatal lesion is accompanied by a spatial memory deficit, this plasticity may compromise function of the remaining circuitry.

Protective role of nerve growth factor against excitatory amino acid injury during neostriatal cholinergic neurons postnatal development.

The interaction between excitatory amino acids (EAAs) and nerve growth factor (NGF) levels were studied on neostriatal cholinergic neurons during postnatal development. Striatal choline acetyltransferase (ChAT) activity and NGF levels were determined 7 days following EAA injection in 7-, 15-, 21-, 30-, and 50-day-old rats. ChAT activity was decreased 7 days after kainate (KA), quinolinate (QUIN), or quisqualate (QUIS) lesion. The reduction was most pronounced in 30-day-old rats. KA injection produced the greatest decrease in ChAT activity. Conversely, KA did not change NGF levels. QUIN and QUIS increased NGF protein and these effects were maximal with lesions in 21-day-old rats. In order to further characterize the effect of EAAs on NGF levels and ChAT activity, the time-course of the lesion was studied. We used 30-day-old rats as the maximal sensitivity of cholinergic neurons to EAAs was observed at this age. ChAT activity decreased 2 days following QUIN or QUIS injection and 1 day after KA. The EAA agonists also changed NGF levels. QUIN induced an increase in NGF levels 1 day after lesion. This effect was maintained to the last time point examined. In contrast, KA and QUIS induced transient increases in NGF levels that were only detected 2 and 4 days after injection, respectively. To study whether NGF is able to regulate EAA excitotoxicity on striatal cholinergic neurons, we studied ChAT activity 7 days after simultaneous injection of NGF plus QUIN, KA, or QUIS. Intrastriatal injection of exogenous NGF was able to block the decrease in ChAT activity observed following EAA injection alone.(ABSTRACT TRUNCATED AT 250 WORDS)