Deficits in coordinated neuronal activity and network topology are striatal hallmarks in Huntington's disease.

BACKGROUND: Network alterations underlying neurodegenerative diseases often precede symptoms and functional deficits. Thus, their early identification is central for improved prognosis. In Huntington's disease (HD), the cortico-striatal networks, involved in motor function processing, are the most compromised neural substrate. However, whether the network alterations are intrinsic of the striatum or the cortex is not fully understood. RESULTS: In order to identify early HD neural deficits, we characterized neuronal ensemble calcium activity and network topology of HD striatal and cortical cultures. We used large-scale calcium imaging combined with activity-based network inference analysis. We extracted collective activity events and inferred the topology of the neuronal network in cortical and striatal primary cultures from wild-type and R6/1 mouse model of HD. Striatal, but not cortical, HD networks displayed lower activity and a lessened ability to integrate information. GABAA receptor blockade in healthy and HD striatal cultures generated similar coordinated ensemble activity and network topology, highlighting that the excitatory component of striatal system is spared in HD. Conversely, NMDA receptor activation increased individual neuronal activity while coordinated activity became highly variable and undefined. Interestingly, by boosting NMDA activity, we rectified striatal HD network alterations. CONCLUSIONS: Overall, our integrative approach highlights striatal defective network integration capacity as a major contributor of basal ganglia dysfunction in HD and suggests that increased excitatory drive may serve as a potential intervention. In addition, our work provides a valuable tool to evaluate in vitro network recovery after treatment intervention in basal ganglia disorders.

Long-term memory deficits in Huntington's disease are associated with reduced CBP histone acetylase activity.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the huntingtin (htt) gene. Although HD is classically considered a motor disorder, there is now considerable evidence that early cognitive deficits appear in patients before the onset of motor disturbances. Here we demonstrate early impairment of long-term spatial and recognition memory in heterozygous HD knock-in mutant mice (Hdh(Q7/Q111)), a genetically accurate HD mouse model. Cognitive deficits are associated with reduced hippocampal expression of CREB-binding protein (CBP) and diminished levels of histone H3 acetylation. In agreement with reduced CBP, the expression of CREB/CBP target genes related to memory, such c-fos, Arc and Nr4a2, was significantly reduced in the hippocampus of Hdh(Q7/Q111) mice compared with wild-type mice. Finally, and consistent with a role of CBP in cognitive impairment in Hdh(Q7/Q111) mice, administration of the histone deacetylase inhibitor trichostatin A rescues recognition memory deficits and transcription of selective CREB/CBP target genes in Hdh(Q7/Q111) mice. These findings demonstrate an important role for CBP in cognitive dysfunction in HD and suggest the use of histone deacetylase inhibitors as a novel therapeutic strategy for the treatment of memory deficits in this disease.

BDNF regulation under GFAP promoter provides engineered astrocytes as a new approach for long-term protection in Huntington's disease.

Brain-derived neurotrophic factor (BDNF) is the main candidate for neuroprotective therapeutic strategies for Huntington's disease. However, the administration system and the control over the dosage are still important problems to be solved. Here we generated transgenic mice overexpressing BDNF under the promoter of the glial fibrillary acidic protein (GFAP) (pGFAP-BDNF mice). These mice are viable and have a normal phenotype. However, intrastriatal administration of quinolinate increased the number of reactive astrocytes and enhanced the release of BDNF in pGFAP-BDNF mice compared with wild-type mice. Coincidentally, pGFAP-BDNF mice are more resistant to quinolinate than wild-type mice, suggesting a protective effect of astrocyte-derived BDNF. To verify this, we next cultured astrocytes from pGFAP-BDNF and wild-type mice for grafting. Wild-type and pGFAP-BDNF-derived astrocytes behave similarly in nonlesioned mice. However, pGFAP-BDNF-derived astrocytes showed higher levels of BDNF and larger neuroprotective effects than the wild-type ones when quinolinate was injected 30 days after grafting. Interestingly, mice grafted with pGFAP-BDNF astrocytes showed important and sustained behavioral improvements over time after quinolinate administration as compared with mice grafted with wild-type astrocytes. These findings show that astrocytes engineered to release BDNF can constitute a therapeutic approach for Huntington's disease.

Brain-derived neurotrophic factor modulates the severity of cognitive alterations induced by mutant huntingtin: involvement of phospholipaseCgamma activity and glutamate receptor expression.

The involvement of brain-derived neurotrophic factor (BDNF) in cognitive processes and the decrease in its expression in Huntington's disease suggest that this neurotrophin may play a role in learning impairment during the disease progression. We therefore analyzed the onset and severity of cognitive deficits in two different mouse models with the same mutant huntingtin but with different levels of BDNF (R6/1 and R6/1:BDNF+/- mice). We observed that BDNF modulates cognitive function in different learning tasks, even before the onset of motor symptoms. R6/1:BDNF+/- mice showed earlier and more accentuated cognitive impairment than R6/1 mice at 5 weeks of age in discrimination learning; at 5 weeks of age in procedural learning; and at 9 weeks of age in alternation learning. At the earliest age at which cognitive impairment was detected, electrophysiological analysis was performed in the hippocampus. All mutant genotypes showed reduced hippocampal long term potentiation (LTP) with respect to wild type but did not show differences between them. Thus, we evaluated the involvement of BDNF-trkB signaling and glutamate receptor expression in the hippocampus of these mice. We observed a decrease in phospholipaseCgamma activity, but not ERK, in R61, BDNF+/- and R6/1:BDNF+/- hippocampus at the age when LTP was altered. However, a specific decrease in the expression of glutamate receptors NR1, NR2A and GluR1 was detected only in R6/1:BDNF+/- hippocampus. Therefore, these results show that BDNF modulates the learning and memory alterations induced by mutant huntingtin. This interaction leads to intracellular changes, such as specific changes in glutamate receptors and in BDNF-trkB signaling through phospholipaseCgamma.

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.

Analysis of antibodies to neuronal surface antigens in adult opsoclonus-myoclonus.

Antibodies against neuronal surface antigens (NSA-ab) have been described in pediatric opsoclonus-myoclonus syndrome (OMS). We analyzed the presence of NSA-ab by flow cytometry and immunocytochemistry of live cerebellar granular neurons (CGN) in the serum of 25 adult patients with idiopathic (14) and paraneoplastic (11) OMS. Paraneoplastic, but not idiopathic, OMS sera showed a CGN surface binding by flow cytometry higher than that of controls (mean MFI (median fluorescence intensity): 29+/-6.9 vs. 20+/-5.8; p=0.001) but only one serum had a binding greater than three standard deviations of controls. OMS sera did not label live CGN by immunocytochemistry. Unlike pediatric OMS, NSA-ab were not detected in adult cases suggesting that the immunity to NSA in OMS is heterogeneous.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Enhances Hippocampal Synaptic Plasticity and Improves Memory Performance in Huntington's Disease.

Deficits in hippocampal synaptic plasticity result in cognitive impairment in Huntington's disease (HD). Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts neuroprotective actions, mainly through the PAC1 receptor. However, the role of PACAP in cognition is poorly understood, and no data exists in the context of Huntington's disease (HD). Here, we investigated the ability of PACAP receptor stimulation to enhance memory development in HD. First, we observed a hippocampal decline of all three PACAP receptor expressions, i.e., PAC1, VPAC1, and VPAC2, in two different HD mouse models, R6/1 and HdhQ7/Q111, from the onset of cognitive dysfunction. In hippocampal post-mortem human samples, we found a specific decrease of PAC1, without changes in VPAC1 and VPAC2 receptors. To determine whether activation of PACAP receptors could contribute to improve memory performance, we conducted daily intranasal administration of PACAP38 to R6/1 mice at the onset of cognitive impairment for seven days. We found that PACAP treatment rescued PAC1 level in R6/1 mice, promoted expression of the hippocampal brain-derived neurotrophic factor, and reduced the formation of mutant huntingtin aggregates. Furthermore, PACAP administration counteracted R6/1 mice memory deficits as analyzed by the novel object recognition test and the T-maze spontaneous alternation task. Importantly, the effect of PACAP on cognitive performance was associated with an increase of VGlut-1 and PSD95 immunolabeling in hippocampus of R6/1 mice. Taken together, these results suggest that PACAP, acting through stimulation of PAC1 receptor, may have a therapeutic potential to counteract cognitive deficits induced in HD.

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.

Effect of glatiramer acetate (Copaxone) on the immunophenotypic and cytokine profile and BDNF production in multiple sclerosis: a longitudinal study.

We assessed the effect of glatiramer acetate (GA) on the immunophenotypic and cytokine profile and the BDNF production by peripheral blood mononuclear cells, and their association with the clinical response in 19 naïve-treated MS patients prospectively followed-up after GA therapy. Two patients withdrew the therapy. After a median follow-up of 21 months, twelve were considered responders and five as non-responders. Non-responder patients had significant longer disease duration and a higher EDSS score at baseline. In the responder group, a significant decrease in the percentage of INF-gamma producing total lymphocytes, CD4+ and CD8+ T cells, and reduced percentage of IL-2 producing CD4+ and CD8+ T cells were observed at 12, 18 and 24 months. These changes were associated with a significant increase in the percentage of CD3+, CD4+ and CD4(+) CD45RA(+) T cells, and BDNF production from month 6 that remained significant throughout the study. We did not observe significant changes in the nonresponder group for any of the parameters studied. Our data suggest that GA treatment induces a downmodulation of proinflammatory cytokines associated with the regulation of the peripheral T cell compartment and with increased production of BDNF that might be related to the clinical response.

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.

Evolution of brain-derived neurotrophic factor levels after autologous hematopietic stem cell transplantation in multiple sclerosis.

A neuroprotective role of inflammation has been suggested based on that immune cells are the main source of brain-derived neurotrophic factor (BDNF). We investigated the 3-year evolution of BDNF levels in serum, CSF and culture supernatant of peripheral blood mononuclear cells (PBMC), unstimulated and stimulated with anti-CD3 and soluble anti-CD28 antibodies, in 14 multiple sclerosis patients who underwent an autologous hematopoietic stem cell transplantation (AHSCT). BDNF levels were correlated with previously reported MRI measures that showed a reduction of T2 lesion load and increased brain atrophy, mainly at first year post-transplant. A significant decrease of serum BDNF levels was seen at 12 months post-transplant. BDNF values were found significantly lower in stimulated but not in unstimulated PBMC supernatants during the follow-up, supporting that AHSCT may induce a down-regulation of BDNF production. The only significant correlation was found between CSF BDNF levels and T2 lesion load before and 1 year after AHSCT, suggesting that BDNF reflects the past and ongoing inflammatory activity and demyelination of these highly active patients. Our study suggests that AHSCT can reduce BDNF levels to values associated with lower activity. This decrease does not seem to correlate with the brain atrophy measures observed in the MRI.

BDNF up-regulates TrkB protein and prevents the death of CA1 neurons following transient forebrain ischemia.

The neurotrophin family of growth factors, which includes Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT4/5) bind and activate specific tyrosine kinase (Trk) receptors to promote cell survival and growth of different cell populations. For these reasons, growing attention has been paid to the use of neurotrophins as therapeutic agents in neurodegeneration, and to the regulation of the expression of their specific receptors by the ligands. BDNF expression, as revealed by immunohistochemistry, is found in the pre-subiculum, CA1, CA3, and dentate gyrus of the hippocampus. Strong TrkB immunoreactivity is present in most CA3 neurons but only in scattered neurons of the CA1 area. Weak TrkB immunoreactivity is found in the granule cell layer of the dentate gyrus. Unilateral grafting of BDNF-transfected fibroblasts into the hippocampus resulted in a marked increase in the intensity of the immunoreaction and in the number of TrkB-immunoreactive neurons in the granule cell layer of the dentate gyrus, pre-subiculum and CA1 area in the vicinity of the graft. No similar effects were produced after the injection of control mock-transfected fibroblasts. Delayed cell death in the CA1 area was produced following 5 min of forebrain ischemia in the gerbil. The majority of living cells in the CA1 area at the fourth day were BDNF/TrkB immunoreactive. Unilateral grafting of control mock-transfected or BDNF fibroblasts two days before ischemia resulted in a moderate non-specific protection of TrkB-negative, but not TrkB-positive cells, in the CA1 area of the grafted side. This finding is in line with a vascular and glial reaction, as revealed, by immunohistochemistry using astroglial and microglial cell markers. This astroglial response was higher in the grafted side than in the contralateral side in ischemic gerbils, but no differences were seen between BDNF-producing and non-BDNF-producing grafts. However, grafting of BDNF-producing fibroblasts two days before ischemia significantly and specifically prevented nerve cells from dying in the CA1 area of the ipsilateral hippocampus. Cell survival was associated with increased TrkB immunoreactivity as the majority of living cells were TrkB immunoreactive. Thus, our results show that BDNF is able to up-regulate the expression of TrkB in control and pathological states, and that BDNF prevention of neuronal death following transient forebrain ischemia is associated with increased expression of its specific receptor.

Bone morphogenetic protein-2, but not bone morphogenetic protein-7, promotes dendritic growth and calbindin phenotype in cultured rat striatal neurons.

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily. They are widely expressed in the mammalian nervous system, where they exert trophic effects on several neuronal populations. We studied the neurotrophic activity of bone morphogenetic protein-2 and bone morphogenetic protein-7 (also called osteogenic protein-1) on cultured striatal cells, previously shown to express bone morphogenetic protein ligands and receptors. Our results indicate that only bone morphogenetic protein-2 promoted the differentiation of GABAergic neurons, especially of the calbindin-positive subpopulation, the subset of projecting striatal neurons that degenerates in Huntington's disease. Bone morphogenetic protein-2 increased the area, perimeter and degree of arborization of GABAergic neurons, promoting calbindin phenotype without altering proliferation or apoptosis. In contrast, neither bone morphogenetic protein-2 nor -7 affected striatal cholinergic interneurons. However, they both increased the number of glial fibrillary acidic protein-positive cells. Suppression of glial proliferation with 5-fluorodeoxyuridine did not abolish bone morphogenetic protein-2 effects on the differentiation of striatal neurons, ruling out an indirect mechanism through astrocytes. In conclusion, our results show that bone morphogenetic protein-2 promotes the differentiation of cultured GABAergic striatal neurons, suggesting that bone morphogenetic proteins are involved in the development of the striatum.

Dopaminergic system mediates only delta-opiate inhibition of endogenous acetylcholine release evoked by glutamate from rat striatal slices.

In order to study the role of the dopaminergic system in the mu- or delta-opioid inhibition of endogenous acetylcholine release evoked by glutamate, we blocked the dopaminergic transmission with dopaminergic antagonists and/or 6-hydroxydopamine lesions. In all these experimental conditions we show that dopaminergic antagonists by themselves could not modify the glutamate-evoked acetylcholine release, and the selective D1 antagonist (SCH 23390) was unable to modify the mu- or delta-opioid inhibition of glutamate-evoked acetylcholine release. However, in the non-lesioned animals and in the contralateral striata to 6-hydroxydopamine lesions, D2 antagonists (haloperidol or sulpiride, 10 microM) prevented the effects of delta-opiate agonists ([D-Ala2, D-Leu5]enkephalin, 1 microM and [D-Pen2, D-Pen5]enkephalin, 0.1 microM), but not the effects of mu-opiate agonists (morphine or [D-Ala2, Gly(ol)5]enkephalin, 1 microM). Furthermore, [D-Ala2, D-Leu5]enkephalin inhibition of glutamate-evoked acetylcholine release was prevented by D2 antagonists in a concentration-dependent manner. Instead, in the 6-hydroxydopamine-lesioned side, while [D-Ala2, D-Leu5]enkephalin (1 microM) inhibition of glutamate-evoked acetylcholine release was completely abolished, morphine (1 microM) inhibition remained unchanged. We conclude that the inhibition of glutamate-evoked endogenous acetylcholine release by delta-opiate agonists, unlike mu-opiate agonists, depends on dopaminergic terminals and D2 receptors. Furthermore, these results suggest that the inhibition by delta-opiate agonists could be the result of dopamine release from dopaminergic terminals and its action on D2 receptors.

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.

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.

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.

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.

Excitatory amino acids release endogenous acetylcholine from rat striatal slices: Regulation by gamma-aminobutyric acid.

Endogenous acetylcholine release from rat striatal slices in response to several excitatory amino acids and its regulation by gamma-aminobutyric acid were investigated using a chemiluminescent reaction to detect this release continuously. The amount of acetylcholine released by glutamate (1 ?M) was 60% of that released by KCl (50 mM). Quisqualic acid and kainic acid, both 1 ?M, elicited only half as much acetylcholine release as did 1 ?M of glutamic acid or N- methyl- d -aspartate . Only the effects of glutamate or N- methyl- d -aspartate were blocked by ?-aminoadipic acid, but not by glutamic acid diethylester, both at 100 ?M. Gamma-aminobutyric acid modulated acetylcholine release evoked by either high potassium or glutamate. However, the inhibitory effect of gamma-aminobutyric acid was greater (54%) when the slices were stimulated by glutamate than when depolarized by KCl (40%). These actions were completely reversed by picrotoxin or bicuculline, both at 100 ?M. It is concluded on the one hand that glutamate elicits acetylcholine release by acting on the N- methyl- d -aspartate receptor. On the other hand, gamma-aminobutyric acid inhibited to a greater extent endogenous acetylcholine release evoked by glutamate than that evoked by potassium. These findings suggest that gamma-aminobutyric acid exerts an important regulation on cholinergic neurons stimulated by the glutamatergic corticostriatal pathway.

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.