The Flavonol Quercitrin Hinders GSK3 Activity and Potentiates the Wnt/ß-Catenin Signaling Pathway.

The Wnt/ß-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer's disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3ß S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-ß oligomers (AßO) in mice. Finally, quercitrin rescues AßO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/ß-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.

Interaction of amyloid-ß (Aß) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice.

Brain accumulation of the amyloid-ß protein (Aß) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aß oligomers (AßOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AßOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AßOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AßOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AßO binding to hippocampal neurons and prevented AßO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AßOs in mice. The results indicate that Nrx2α and NL1 are targets of AßOs and that prevention of this interaction reduces the deleterious impact of AßOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AßOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.

Inflammatory demyelination alters subcortical visual circuits.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory demyelinating disease classically associated with axonal damage and loss; more recently, however, synaptic changes have been recognized as additional contributing factors. An anatomical area commonly affected in MS is the visual pathway; yet, changes other than those associated with inflammatory demyelination of the optic nerve, i.e., optic neuritis, have not been described in detail. METHODS: Adult mice were subjected to a diet containing cuprizone to mimic certain aspects of inflammatory demyelination as seen in MS. Demyelination and inflammation were assessed by real-time polymerase chain reaction and immunohistochemistry. Synaptic changes associated with inflammatory demyelination in the dorsal lateral geniculate nucleus (dLGN) were determined by immunohistochemistry, Western blot analysis, and electrophysiological field potential recordings. RESULTS: In the cuprizone model, demyelination was observed in retinorecipient regions of the subcortical visual system, in particular the dLGN, where it was found accompanied by microglia activation and astrogliosis. In contrast, anterior parts of the pathway, i.e., the optic nerve and tract, appeared largely unaffected. Under the inflammatory demyelinating conditions, as seen in the dLGN of cuprizone-treated mice, there was an overall decrease in excitatory synaptic inputs from retinal ganglion cells. At the same time, the number of synaptic complexes arising from gamma-aminobutyric acid (GABA)-generating inhibitory neurons was found increased, as were the synapses that contain the N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B and converge onto inhibitory neurons. These synaptic changes were functionally found associated with a shift toward an overall increase in network inhibition. CONCLUSIONS: Using the cuprizone model of inflammatory demyelination, our data reveal a novel form of synaptic (mal)adaption in the CNS that is characterized by a shift of the excitation/inhibition balance toward inhibitory network activity associated with an increase in GABAergic inhibitory synapses and a possible increase in excitatory input onto inhibitory interneurons. In addition, our data recognize the cuprizone model as a suitable tool in which to assess the effects of inflammatory demyelination on subcortical retinorecipient regions of the visual system, such as the dLGN, in the absence of overt optic neuritis.

Cognitive dysfunction is sustained after rescue therapy in experimental cerebral malaria, and is reduced by additive antioxidant therapy.

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.

Thyroid hormone induces cerebellar neuronal migration and Bergmann glia differentiation through epidermal growth factor/mitogen-activated protein kinase pathway.

Cerebellar development in the postnatal period is mainly characterized by an intense cellular proliferation in the external granular layer, followed by migration of granular cells in the molecular layer along the Bergmann glia (BG) fibers. Cerebellar ontogenesis undergoes dramatic modulation by thyroid hormones (THs), although their mechanism of action in this organ is still largely unknown. We previously demonstrated that THs induce astrocytes to secrete epidermal growth factor (EGF), which thus promotes cerebellar neuronal proliferation and extracellular matrix remodeling in vitro. In the present study, we investigated the effect of the TH/EGF pathway on granule neuronal migration. By taking advantage of rat explant and dissociated culture assays, we showed that cerebellar astrocytes treated with TH promote granule cell migration. The addition of neutralizing antibodies against EGF or the pharmacological inhibitor of EGF signaling, bis-tyrphostin, completely inhibited TH-astrocyte-induced migration. Likewise, the addition of EGF itself greatly increased neuronal migration. Treatment of BG-dissociated cultures by EGF dramatically induced an alteration in cell morphology, characterized by an elongation in the glial process. Both neuronal migration and BG elongation were inhibited by the mitogen-activated protein kinase pathway inhibitor PD98059, suggesting that these events might be associated. Together, our results suggest that, by inducing EGF secretion, THs promote neuronal migration through BG elongation. Our data provide new clues to the molecular mechanism of THs in cerebellar development, and may contribute to a better understanding of some neuroendocrine disorders associated with migration deficits.

Radial Glia-endothelial Cells' Bidirectional Interactions Control Vascular Maturation and Astrocyte Differentiation: Impact for Blood-brain Barrier Formation.

BACKGROUND: In the developing cerebral cortex, Radial Glia (RG) multipotent neural stem cell, among other functions, differentiate into astrocytes and serve as a scaffold for blood vessel development. After some time, blood vessel Endothelial Cells (ECs) become associated with astrocytes to form the neurovascular Blood-Brain Barrier (BBB) unit. OBJECTIVE: Since little is known about the mechanisms underlying bidirectional RG-ECs interactions in both vascular development and astrocyte differentiation, this study investigated the impact of interactions between RG and ECs mediated by secreted factors on EC maturation and gliogenesis control. METHODS: First, we demonstrated that immature vasculature in the murine embryonic cerebral cortex physically interacts with Nestin positive RG neural stem cells in vivo. Isolated Microcapillary Brain Endothelial Cells (MBEC) treated with the conditioned medium from RG cultures (RG-CM) displayed decreased proliferation, reduction in the protein levels of the endothelial tip cell marker Delta-like 4 (Dll4), and decreased expression levels of the vascular permeability associated gene, plasmalemma vesicle-associated protein-1 (PLVAP1). These events were also accompanied by increased levels of the tight junction protein expression, zonula occludens-1 (ZO-1). RESULTS: Finally, we demonstrated that isolated RG cells cultures treated with MBEC conditioned medium promoted the differentiation of astrocytes in a Vascular Endothelial Growth Factor-A (VEGF-A) dependent manner. CONCLUSION: These results suggest that the bidirectional interaction between RG and ECs is essential to induce vascular maturation and astrocyte generation, which may be an essential cell-cell communication mechanism to promote BBB establishment.

Detrimental Effects of HMGB-1 Require Microglial-Astroglial Interaction: Implications for the Status Epilepticus -Induced Neuroinflammation.

Temporal Lobe Epilepsy (TLE) is the most common form of human epilepsy and available treatments with antiepileptic drugs are not disease-modifying therapies. The neuroinflammation, neuronal death and exacerbated plasticity that occur during the silent period, following the initial precipitating event (IPE), seem to be crucial for epileptogenesis. Damage Associated Molecular Patterns (DAMP) such as HMGB-1, are released early during this period concomitantly with a phenomenon of reactive gliosis and neurodegeneration. Here, using a combination of primary neuronal and glial cell cultures, we show that exposure to HMGB-1 induces dendrite loss and neurodegeneration in a glial-dependent manner. In glial cells, loss of function studies showed that HMGB-1 exposure induces NF-κB activation by engaging a signaling pathway that involves TLR2, TLR4, and RAGE. In the absence of glial cells, HMGB-1 failed to induce neurodegeneration of primary cultured cortical neurons. Moreover, purified astrocytes were unable to fully respond to HMGB-1 with NF-κB activation and required microglial cooperation. In agreement, in vivo HMGB-1 blockage with glycyrrhizin, immediately after pilocarpine-induced status epilepticus (SE), reduced neuronal degeneration, reactive astrogliosis and microgliosis in the long term. We conclude that microglial-astroglial cooperation is required for astrocytes to respond to HMGB-1 and to induce neurodegeneration. Disruption of this HMGB-1 mediated signaling pathway shows beneficial effects by reducing neuroinflammation and neurodegeneration after SE. Thus, early treatment strategies during the latency period aimed at blocking downstream signaling pathways activated by HMGB-1 are likely to have a significant effect in the neuroinflammation and neurodegeneration that are proposed as key factors in epileptogenesis.

Neonatal infection leads to increased susceptibility to Aß oligomer-induced brain inflammation, synapse loss and cognitive impairment in mice.

Harmful environmental stimuli during critical stages of development can profoundly affect behavior and susceptibility to diseases. Alzheimer disease (AD) is the most frequent neurodegenerative disease, and evidence suggest that inflammatory conditions act cumulatively, contributing to disease onset. Here we investigated whether infection early in life can contribute to synapse damage and cognitive impairment induced by amyloid-ß oligomers (AßOs), neurotoxins found in AD brains. To this end, wild-type mice were subjected to neonatal (post-natal day 4) infection by Escherichia coli (1 × 104 CFU/g), the main cause of infection in low-birth-weight premature infants in the US. E. coli infection caused a transient inflammatory response in the mouse brain starting shortly after infection. Although infected mice performed normally in behavioral tasks in adulthood, they showed increased susceptibility to synapse damage and memory impairment induced by low doses of AßOs (1 pmol; intracerebroventricular) in the novel object recognition paradigm. Using in vitro and in vivo approaches, we show that microglial cells from E. coli-infected mice undergo exacerbated activation when exposed to low doses of AßOs. In addition, treatment of infected pups with minocycline, an antibiotic that inhibits microglial pro-inflammatory polarization, normalized microglial response to AßOs and restored normal susceptibility of mice to oligomer-induced cognitive impairment. Interestingly, mice infected with by E. coli (1 × 104 CFU/g) during adolescence (post-natal day 21) or adulthood (post-natal day 60) showed normal cognitive performance even in the presence of AßOs (1 pmol), suggesting that only infections at critical stages of development may lead to increased susceptibility to amyloid-ß-induced toxicity. Altogether, our findings suggest that neonatal infections can modulate microglial response to AßOs into adulthood, thus contributing to amyloid-ß-induced synapse damage and cognitive impairment.

Cyclic AMP enhancers and Abeta oligomerization blockers as potential therapeutic agents in Alzheimer's disease.

One of the earliest manifestations of Alzheimer's disease (AD) is the characteristic inability of affected individuals to form new memories. Memory impairment appears to significantly predate the death of nerve cells, implying that neuronal dysfunction is responsible for the pathophysiology of early stage AD. Mounting evidence now indicates that soluble oligomers of the amyloid-beta peptide (Abeta) are the main neurotoxins that lead to early neuronal dysfunction and memory deficits in AD. Cyclic AMP (cAMP) is a central component of intracellular signaling pathways that regulate a wide range of biological functions, including memory. Among other actions, cAMP triggers the phosphorylation and activation of the cAMP responsive element binding protein (CREB), a transcription factor that regulates the expression of genes that are important for long-term memory. Here, we discuss recent evidence suggesting that cAMP enhancing compounds may find applications as neurocognitive enhancers in AD and in other neurological disorders, as well as possible roles of cAMP in the regulation of neuronal regeneration. In particular, we review recent results showing that low concentrations of 2,4-dinitrophenol (DNP) upregulate neuronal cAMP and tau levels, promote neurite outgrowth and neuronal differentiation and block the oligomerization and neurotoxicity of Abeta. Possible implications of these findings in the development of novel therapeutic approaches in AD are discussed.

Neuroprotective astrocyte-derived insulin/insulin-like growth factor 1 stimulates endocytic processing and extracellular release of neuron-bound Aß oligomers.

Synaptopathy underlying memory deficits in Alzheimer's disease (AD) is increasingly thought to be instigated by toxic oligomers of the amyloid beta peptide (AßOs). Given the long latency and incomplete penetrance of AD dementia with respect to Aß pathology, we hypothesized that factors present in the CNS may physiologically protect neurons from the deleterious impact of AßOs. Here we employed physically separated neuron-astrocyte cocultures to investigate potential non-cell autonomous neuroprotective factors influencing AßO toxicity. Neurons cultivated in the absence of an astrocyte feeder layer showed abundant AßO binding to dendritic processes and associated synapse deterioration. In contrast, neurons in the presence of astrocytes showed markedly reduced AßO binding and synaptopathy. Results identified the protective factors released by astrocytes as insulin and insulin-like growth factor-1 (IGF1). The protective mechanism involved release of newly bound AßOs into the extracellular medium dependent upon trafficking that was sensitive to exosome pathway inhibitors. Delaying insulin treatment led to AßO binding that was no longer releasable. The neuroprotective potential of astrocytes was itself sensitive to chronic AßO exposure, which reduced insulin/IGF1 expression. Our findings support the idea that physiological protection against synaptotoxic AßOs can be mediated by astrocyte-derived insulin/IGF1, but that this protection itself is vulnerable to AßO buildup.

Brain infusion of α-synuclein oligomers induces motor and non-motor Parkinson's disease-like symptoms in mice.

Parkinson's disease (PD) is characterized by motor dysfunction, which is preceded by a number of non-motor symptoms including olfactory deficits. Aggregation of α-synuclein (α-syn) gives rise to Lewy bodies in dopaminergic neurons and is thought to play a central role in PD pathology. However, whether amyloid fibrils or soluble oligomers of α-syn are the main neurotoxic species in PD remains controversial. Here, we performed a single intracerebroventricular (i.c.v.) infusion of α-syn oligomers (α-SYOs) in mice and evaluated motor and non-motor symptoms. Familiar bedding and vanillin essence discrimination tasks showed that α-SYOs impaired olfactory performance of mice, and decreased TH and dopamine levels in the olfactory bulb early after infusion. The olfactory deficit persisted until 45days post-infusion (dpi). α- SYO-infused mice behaved normally in the object recognition and forced swim tests, but showed increased anxiety-like behavior in the open field and elevated plus maze tests 20 dpi. Finally, administration of α-SYOs induced late motor impairment in the pole test and rotarod paradigms, along with reduced TH and dopamine content in the caudate putamen, 45 dpi. Reduced number of TH-positive cells was also seen in the substantia nigra of α-SYO-injected mice compared to control. In conclusion, i.c.v. infusion of α-SYOs recapitulated some of PD-associated non-motor symptoms, such as increased anxiety and olfactory dysfunction, but failed to recapitulate memory impairment and depressive-like behavior typical of the disease. Moreover, α-SYOs i.c.v. administration induced motor deficits and loss of TH and dopamine levels, key features of PD. Results point to α-syn oligomers as the proximal neurotoxins responsible for early non-motor and motor deficits in PD and suggest that the i.c.v. infusion model characterized here may comprise a useful tool for identification of PD novel therapeutic targets and drug screening.

Neuritogenesis and neuronal differentiation promoted by 2,4-dinitrophenol, a novel anti-amyloidogenic compound.

Neurite outgrowth is a critical event in neuronal development, formation, and remodeling of synapses, response to injury, and regeneration. We examined the effects of 2,4-dinitrophenol (DNP), a recently described blocker of the aggregation and neurotoxicity of the beta-amyloid peptide, on neurite elongation of central neurons. Morphometric analysis of rat embryo hippocampal and cortical neuronal cultures showed that neurite outgrowth was stimulated by DNP. This effect was accompanied by increases in the neuronal levels of the microtubule-associated protein tau and of cyclic adenosine 3',5' monophosphate (cAMP). DNP also promoted cAMP accumulation, increased tau level, neurite outgrowth, and neuronal differentiation in the mouse neuroblastoma cell line N2A. We show that DNP-induced differentiation requires activation of the extracellular signal-regulated kinase (ERK). The finding that DNP promotes neuritogenesis and neuronal differentiation suggests that, in addition to its anti-amyloidogenic actions, it may be a useful lead compound in the development of novel therapeutic approaches targeting neurite dystrophy and synaptic dysfunction in neurodegenerative pathologies such as Alzheimer's disease.

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation.

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-ß family, TGF-beta 1 (TGF-ß1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-ß1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-ß1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-ß1 and its receptor, TßRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1-P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-ß receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-ß1. Treatment of granular cell cultures with TGF-ß1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TßRI activation because addition of a pharmacological inhibitor of TGF-ß, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-ß1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons.

Corrigendum: TGF-ß1 promotes cerebral cortex radial glia-astrocyte differentiation in vivo.

[This corrects the article on p. 393 in vol. 8, PMID: 25484855.].

Thyroid hormone treated astrocytes induce maturation of cerebral cortical neurons through modulation of proteoglycan levels.

Proper brain neuronal circuitry formation and synapse development is dependent on specific cues, either genetic or epigenetic, provided by the surrounding neural environment. Within these signals, thyroid hormones (T3 and T4) play crucial role in several steps of brain morphogenesis including proliferation of progenitor cells, neuronal differentiation, maturation, migration, and synapse formation. The lack of thyroid hormones during childhood is associated with several impair neuronal connections, cognitive deficits, and mental disorders. Many of the thyroid hormones effects are mediated by astrocytes, although the mechanisms underlying these events are still unknown. In this work, we investigated the effect of 3, 5, 3'-triiodothyronine-treated (T3-treated) astrocytes on cerebral cortex neuronal differentiation. Culture of neural progenitors from embryonic cerebral cortex mice onto T3-treated astrocyte monolayers yielded an increment in neuronal population, followed by enhancement of neuronal maturation, arborization and neurite outgrowth. In addition, real time PCR assays revealed an increase in the levels of the heparan sulfate proteoglycans, Glypican 1 (GPC-1) and Syndecans 3 e 4 (SDC-3 e SDC-4), followed by a decrease in the levels of the chondroitin sulfate proteoglycan, Versican. Disruption of glycosaminoglycan chains by chondroitinase AC or heparanase III completely abolished the effects of T3-treated astrocytes on neuronal morphogenesis. Our work provides evidence that astrocytes are key mediators of T3 actions on cerebral cortex neuronal development and identified potential molecules and pathways involved in neurite extension; which might eventually contribute to a better understanding of axonal regeneration, synapse formation, and neuronal circuitry recover.

Structure of laminin substrate modulates cellular signaling for neuritogenesis.

Laminin, a major component of basement membranes, can self-assemble in vitro into a typical mesh-like structure, according to a mass-action-driven process. Previously, we showed that pH acidification dramatically increased the efficiency of laminin self-assembly, practically abolishing the necessity for a minimal protein concentration. Here we have characterized the morphologies of laminin matrices produced in either neutral or acidic conditions and compared their capacities to induce neuritogenesis of rat embryonic cortical neurons. Although laminin matrices formed in neutral buffer presented aggregates of heterogeneous morphology, the acidic matrix consisted of a homogeneous hexagonal sheet-like structure. The latter was comparable to the matrix assembled in vivo at the inner limiting membrane of the retina in newborn rats, shown here, and to matrices secreted by cultivated cells, shown elsewhere. The average neurite length of cortical neurons plated on acidic matrices was 244.9 micro m, whereas on neutral matrices this value dropped to 104.1 micro m. Increased neuritogenesis on the acidic matrix seemed to be associated with a higher degree of neuronal differentiation, since cell proliferation was immediately arrested upon plating, whereas on neutral matrices, the cell number increased six-fold within 24 hours. Investigation of the mechanisms mediating neurite outgrowth on each condition revealed that the extensive neuritogenesis observed on the acidic matrix involved activation of protein kinase A, whereas moderate neuritogenesis on neutral laminin was mediated by activation of protein kinase C and/or myosin light-chain kinase. Explants of cerebral cortex from P2 rats did not grow on the neutral laminin substrate but presented extensive cell migration and neurite outgrowth on the acidic laminin matrix. We propose that laminin can self-assemble independently of cell contact and that the assembling mode differentially modulates neuritogenesis and neuroplasticity.

Sialic acid residues on astrocytes regulate neuritogenesis by controlling the assembly of laminin matrices.

In the developing nervous system migrating neurons and growing axons are guided by diffusible and/or substrate-bound cues, such as extracellular matrix-associated laminin. In a previous work we demonstrated that laminin molecules could self-assemble in two different manners, giving rise to matrices that could favor either neuritogenesis or proliferation of cortical precursor cells. We investigated whether the ability of astrocytes to promote neuritogenesis of co-cultivated neurons was modulated by the assembling mode of the laminin matrix secreted by them. We compared the morphologies and neuritogenic potentials of laminin deposited by in vitro-differentiated astrocytes obtained from embryonic or neonatal rat brain cortices. We showed that, while permissive astrocytes derived from embryonic brain produced a flat laminin matrix that remained associated to the cell surface, astrocytes derived from newborn brain secreted a laminin matrix resembling a fibrillar web that protruded from the cell plane. The average neurite lengths obtained for E16 neurons cultured on each astrocyte layer were 198+/-22 and 123+/-13 microm, respectively. Analyses of surface-associated electrostatic potentials revealed that embryonic astrocytes presented a pI of -2.8, while in newborn cells this value was -3.8. Removal of the sialic acid groups on the embryonic monolayer by neuraminidase treatment led to the immediate release of matrix-associated laminin. Interestingly, laminin reassembled 1 hour after neuraminidase removal converted to the features of the newborn matrix. Alternatively, treatment of astrocytes with the cholesterol-solubilizing detergent methyl-beta-cyclodextrin also resulted in release of the extracellular laminin. To test the hypothesis that sialic-acid-containing lipids localized at cholesterol-rich membrane domains could affect the process of laminin assembly, we devised a cell-free assay where laminin polymerization was carried out over artificial lipid films. Films of either a mixture of gangliosides or pure ganglioside GT1b induced formation of matrices of morpho-functional features similar to the matrices deposited by embryonic astrocytes. Conversely, films of phosphatidylcholine or ganglioside GM1 led to the formation of bulky laminin aggregates that lacked a defined structure. We propose that the expression of negative lipids on astrocytes can control the extracellular polymerization of laminin and, consequently, the permissivity to neuritogenesis of astrocytes during development.