Activation of cannabinoid type 1 receptor (CB1) modulates oligodendroglial process branching complexity in rat hippocampal cultures stimulated by olfactory ensheathing glia-conditioned medium.

The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.

An Essential Role for Alzheimer's-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis.

Human amyloid beta peptide (Aß) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AßOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AßO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aß as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AßOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AßO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AßOs, are linked to Alzheimer's disease (AD). To test whether the AßOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AßO-selective antibody to prematurely lower the levels of AßOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AßOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.

A novel crosslinking protocol stabilizes amyloid ß oligomers capable of inducing Alzheimer's-associated pathologies.

Amyloid ß oligomers (AßOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AßO-targeting diagnostics and therapeutics, the AßO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AßOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aß in a soluble oligomeric conformation. With DFDNB, solutions of Aß that would otherwise convert to large aggregates instead yield solutions of stable AßOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AßOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AßO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AßOs in structure-function studies.

Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System.

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.

Neuro-glial cannabinoid receptors modulate signaling in the embryonic avian retina.

Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l-Aspartate (L-ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212-2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]-GABA release induced by KCl or L-ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation.

Prion Protein Modulates Monoaminergic Systems and Depressive-like Behavior in Mice.

We sought to examine interactions of the prion protein (PrP(C)) with monoaminergic systems due to: the role of PrP(C) in both Prion and Alzheimer diseases, which include clinical depression among their symptoms, the implication of monoamines in depression, and the hypothesis that PrP(C) serves as a scaffold for signaling systems. To that effect we compared both behavior and monoaminergic markers in wild type (WT) and PrP(C)-null (PrP(-/-)) mice. PrP(-/-) mice performed poorly when compared with WT in forced swimming, tail suspension, and novelty suppressed feeding tests, typical of depressive-like behavior, but not in the control open field nor rotarod motor tests; cyclic AMP responses to stimulation of D1 receptors by dopamine was selectively impaired in PrP(-/-) mice, and responses to serotonin, but not to norepinephrine, also differed between genotypes. Contents of dopamine, tyrosine hydroxylase, and the 5-HT5A serotonin receptor were increased in the cerebral cortex of PrP(-/-), as compared with WT mice. Microscopic colocalization, as well as binding in overlay assays were found of PrP(C) with both the 5HT5A and D1, but not D4 receptors. The data are consistent with the scaffolding of monoaminergic signaling modules by PrP(C), and may help understand the pathogenesis of clinical depression and neurodegenerative disorders.

Exogenous ß-amyloid peptide interferes with GLUT4 localization in neurons.

Aging represents a major risk factor for numerous illnesses that are of increasing importance to society, including two of the most prevalent: diabetes and Alzheimer's disease. Studies have shown that diabetes is a risk factor for spontaneous Alzheimer's disease. While these studies suggest that diabetes can contribute to Alzheimer's disease, the implications of AD on diabetes are practically unexplored. The major mediator of the pathophysiological effects, the Aß42 peptide, has been shown to enter neurons and lead to an alteration of the intracellular distribution of the molecular motor myosin Vb. Myosin Vb functions in memory and learning by participating in the strengthening of the long-term potentiation (LTP) of synaptic transmissions. It has also been implicated in the translocation of the glucose transporter, GLUT4, to the plasma membrane in response to insulin, a process that is defective in diabetes. Here, the effect on GLUT4 upon entry of the Aß42 peptide into cultured chick retinal neurons was explored. The results suggest an alteration in distribution and a reduced level at the cell surface, as well as an increased colocalization with myosin Vb, which can partially explain the changes in glucose metabolism associated with AD. It is also shown that the presence of the Aß40 peptide inhibits the internalization of the Aß42 peptide in cultured cells. Together, the results provide additional targets for the development of therapeutics against the progression and effects of Alzheimer's disease.

Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input.

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease.

Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.

Pituitary adenylyl cyclase-activating polypeptide receptor re-sensitization induces plastic changes in the dopaminergic phenotype in the mature avian retina.

Pituitary Adenylyl Cyclase-Activating Polypeptide (PACAP) is a neuroactive peptide present in the avian retina where it activates adenylyl cyclase (AC) since early in development via PACAP receptors. The synthesis of cAMP in response to PACAP is observed since embryonic day 8/9 (E8/9). After E12, signaling via PACAP receptors desensitizes, reaching very low levels in the mature tissue. We show here that chronic administration of PACAP in vitro desensitizes PACAP-induced cAMP accumulation, while the administration of the PACAP antagonist (PACAP 6-38) re-sensitizes PACAP receptor/cyclase system in vitro and in vivo. Moreover, a twofold increase in the number of tyrosine hydroxylase positive (TH⁺) cells is observed after in vivo injection of PACAP6-38. NURR1, a transcription factor associated with the differentiation of dopaminergic cells in the CNS, is present in the chick retina in all developmental stages studied. The presence of NURR1 positive cells in the mature tissue far exceeds the number of TH⁺ cells, suggesting that these NURR1-positive cells might have the potential to express the dopaminergic phenotype. Our data show that if PACAP signaling is increased in mature retinas, plastic changes in dopaminergic phenotype can be achieved.

Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-ß peptide oligomers.

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-ß peptide (Aß). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aß oligomers (AßOs). AßOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AßOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AßOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AßOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AßO binding sites, fully prevented AßO-induced inhibition of ChAT. Interestingly, we found that AßOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AßO-targeted neurons. Reduction in ChAT activity instigated by AßOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.

Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures.

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 µm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.

ß-Amyloid peptide is internalized into chick retinal neurons and alters the distribution of myosin Vb.

The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of ß-amyloid (Aß) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that Aß also accumulates within neurons and this intraneuronal Aß accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here, we report on the internalization of a fluorescently labeled Aß peptide into cultured chick retinal neurons. The pattern of Aß distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized Aß peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aß production and Aß clearance, suggesting a role for myosin Vb in this process.

Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse.

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-ß peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Exchange of extracellular L-glutamate by intracellular D-aspartate: the main mechanism of D-aspartate release in the avian retina.

D-aspartate is present in significant concentrations throughout the nervous tissue but its physiological role is still under discussion. Here, we report the process of d-aspartate release in retinal cells. [(3)H]-d-aspartate release occurs through a glutamate/aspartate exchange mechanism using excitatory amino acid transporters. This process is sodium-dependent and it is not prevented by glutamate receptor antagonists such as MK-801, DNQX or AIDA nor mimicked by glutamatergic agonists like kainate, NMDA or trans-ACPD. In vitro experiments indicate that the great majority of d-aspartate release is performed by neuronal cells and to a much lower extent by glial cells. This glutamate-mediated release process is mimicked by the competitive glutamate transporter antagonist l-trans-PDC and inhibited by the non-competitive transporter antagonist TBOA. Instead of the classical calcium-dependent exocytosis or transporter-reversal mediated neuronal release, d-aspartate efflux in the retina occurs mostly, if not exclusively, via an exchange of external l-glutamate by d-aspartate predominantly present in the cytoplasmatic compartment of neurons. These data also suggest that this process narrows down the specificity of excitatory signaling in the microenvironment of the synapses, reinforcing NMDA receptor activation by d-aspartate at the cost of reduction in the overall activation of excitatory amino acid receptors promoted by l-glutamate.

Amyloid-ß decreases nitric oxide production in cultured retinal neurons: a possible mechanism for synaptic dysfunction in Alzheimer's disease?

The neurotoxicity of the amyloid-ß peptide (Aß) appears to be, at least in part, related to pathological activation of glutamate receptors by Aß aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer's disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aß42 and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that L-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aß42 (20 µM) or glutamate (1 mM). Surprisingly, at short incubation times both Aß and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aß and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aß or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aß and glutamate suggests a possible mechanism leading to synaptic failure in AD.

Modulation of A1 adenosine receptor expression by cell aggregation and long-term activation of A2a receptors in cultures of avian retinal cells: involvement of the cyclic AMP/PKA pathway.

The expression of A1 and A2a adenosine receptors is developmentally regulated in the chick retina, but little is known about the factors important for this regulation. Here, we show that cell aggregation and cAMP analogs promote a dramatic increase in A1 receptor expression. Importantly, a long-term stimulation of A2a receptors also promotes an increase of A1 receptor expression accompanied by a down-regulation of A2a receptors. Chick embryo retina cultures grown in the form of aggregates or dispersed cells accumulate cAMP when stimulated with dopamine or the adenosine agonist 2-chloroadenosine. However, inhibition of dopamine-dependent cAMP accumulation by 2-chloroadenosine was observed in aggregate cultures but not in dispersed cell cultures. Accordingly, A1 receptor binding sites were detected in aggregate cultures, but were low or absent from dispersed cell cultures. Interestingly, an increase of A1 binding sites was detected when dispersed cell cultures were treated for 5 days with permeable cAMP analogs, the adenylyl cyclase activator forskolin or A2a receptor agonists. Although a significant amount of A1 receptor protein was detected in dispersed cell cultures by western blot or immunocytochemistry, the long-term stimulation of A2a receptors also promoted an increase of the A1 receptor protein and mRNA, indicating that A2a receptors and cAMP were regulating transcription and/or translation of A1 receptors. We also found an increase of A1 receptors in locations in or near the membrane after treatment with A2a agonist. The long-term stimulation of retinal explants with A2a agonist also promoted an increase of A1 receptor protein. The results indicate that A2a receptors and the cAMP-dependent protein kinase pathway are involved in the regulation of A1 receptor expression during retinal development.

The putrescine analogue 1,4-diamino-2-butanone affects polyamine synthesis, transport, ultrastructure and intracellular survival in Leishmania amazonensis.

Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.