Ana Velasco

Neuronal differentiation is triggered by oleic acid synthesized and released by astrocytes

Unlike in the adult brain, the newborn brain specifically takes up serum albumin during the postnatal period, coinciding with the stage of maximal brain development. Here we report that albumin stimulates oleic acid synthesis by astrocytes from the main metabolic substrates available during brain development. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. Oleic acid promotes axonal growth, neuronal clustering, and expression of the axonal growth‐associated protein‐43, GAP‐43; all these observations indicating neuronal differentiation. The effect of oleic acid on GAP‐43 synthesis is brought about by the activation of protein kinase C, since it was prevented by inhibitors of this kinase, such as H‐7, polymyxin or sphingosine. The expression of GAP‐43 was significantly increased in neurons co‐cultured with astrocytes by the presence of albumin indicating that neuronal differentiation takes place in the presence of oleic acid synthesized and released by astrocytes in situ. In conclusion, during brain development the presence of albumin could play an important role by triggering the synthesis and release of oleic acid by astrocytes, which induces neuronal differentiation.

The neurotrophic effect of oleic acid includes dendritic differentiation and the expression of the neuronal basic helix-loop-helix transcription factor NeuroD2

We have shown recently that the presence of albumin in astrocytes triggers the synthesis and release of oleic acid, which behaves as a neurotrophic factor for neurons. Thus, oleic acid promotes axonal growth together with the expression of the axonal growth‐associated protein, GAP‐43. Here we attempted to elucidate whether the neurotrophic effect of oleic acid includes dendritic differentiation. Our results indicate that oleic acid induces the expression of microtubule associated protein‐2 (MAP‐2), a marker of dendritic differentiation. In addition, the presence of oleic acid promotes the translocation of MAP‐2 from the soma to the dendrites. The time course of MAP‐2 expression during brain development coincides with that of stearoyl‐CoA desaturase, the limiting enzyme of oleic acid synthesis, indicating that both phenomena coincide during development. The effect of oleic acid on MAP‐2 expression is most probably independent of autocrine factors synthesized by neurons because this effect was also observed at low cellular densities. As oleic acid is an activator of protein kinase C, the possible participation of this transduction pathway was studied. Our results indicate that added oleic acid or oleic acid endogenously synthesized by astrocytes exerts its neurotrophic effect through a protein kinase C‐dependent mechanism as the effect was inhibited by sphingosine or two myristoylated peptide inhibitors of protein kinase C. The transduction pathway by which oleic acid induces the expression of genes responsible for neuronal differentiation appears to be mediated by the transcription factor NeuroD2, a regulator of terminal neuronal differentiation.

Megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid

We have previously shown that the uptake and transcytosis of albumin in astrocytes promote the synthesis of the neurotrophic factor oleic acid. Although the mechanism by which albumin induces oleic acid synthesis is well known, the mechanism of albumin uptake in astrocytes remains unknown. In this work, we found that astrocytes express megalin, an endocytic receptor for multiple ligands including albumin. In addition, when the activity of megalin is blocked by specific antibodies or by silencing megalin with specific siRNA, albumin binding and internalization is strongly reduced indicating that megalin is required for albumin binding and internalization in the astrocyte. Since the uptake of albumin in astrocytes aims at synthesizing the neurotrophic factor oleic acid, we tested the ability of megalin‐silenced astrocytes to synthesize and release oleic acid in the presence of albumin. Our results showed that the amount of oleic acid found in the extracellular medium of megalin‐silenced astrocytes was strongly reduced as compared with their controls. Together, the results of this work indicate that megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid. Consequently, the possible involvement of albumin in the holoprosencephalic syndrome observed in megalin‐deficient mice is suggested.

Role of oleic acid as a neurotrophic factor is supported in vivo by the expression of GAP-43 subsequent to the activation of SREBP-1 and the up-regulation of stearoyl-CoA desaturase during postnatal development of the brain

We have recently reported that albumin, a serum protein present in the developing brain, stimulates the synthesis of oleic acid by cultured astrocytes by inducing stearoyl-CoA 9-desaturase, the rate-limiting enzyme in oleic acid synthesis, through activation of the sterol regulatory element-binding protein-1. In this work, we offer evidence supporting the in vivo occurrence of this process during the postnatal development of the rat brain. Our results show that albumin reaches maximal brain level by day 1 after birth, coinciding with activation of the sterol response element binding protein-1, which is responsible for the transcription of the enzymes required for oleic acid synthesis. In addition, the developmental profile of stearoyl-CoA 9-desaturase-1 mRNA expression follows that of sterol regulatory element-binding protein-1 activation, indicating that these phenomena are tightly linked. In a previous work, we showed that oleic acid induces neuronal differentiation, as indicated by the expression of growth associated protein-43. Here, we report that the expression of growth associated protein-43 mRNA peaks at about day 7 after birth, following the maximal expression of stearoyl-CoA 9-desaturase-1 mRNA that occurs between days 3 and 5 postnatally. In conclusion, our results support the hypothesis that the synthesis of oleic acid is linked to neuronal differentiation during rat brain development.

ATP-Sensitive Potassium Channel Regulates Astrocytic Gap Junction Permeability by a Ca2+-Independent Mechanism

Using the scrape‐loading technique in cultured astrocytes, we show that sulfonylureas such as tolbutamide and glybenzcyclamide, which inhibit the ATP‐sensitive K+ channel, prevent the inhibition of gap junction permeability caused by several structurally unrelated uncouplers such as oleic acid, arachidonic acid, endothelin‐1, octanol, and α‐glycyrrhetinic acid. When the intracellular level of Ca2+ was diminished, all the uncouplers tested were still able to inhibit gap junction communication, indicating that their inhibitory effect was not mediated by Ca2+. In addition, tolbutamide and glybenzcyclamide prevented the inhibitory effect of these uncouplers in Ca2+‐depleted astrocytes, suggesting that the inhibition of the ATP‐sensitive K+ channel increases gap junction permeability through a Ca2+‐independent mechanism. The activation of the ATP‐sensitive K+ channel caused by potassium channel openers such as diazoxide and pinacidil led to the inhibition of gap junction communication and overcame the effect of sulfonylureas. These results suggest that the ATP‐sensitive K+ channel regulates gap junctional permeability.

Aberrant Co-localization of Synaptic Proteins Promoted by Alzheimer's Disease Amyloid-β Peptides: Protective Effect of Human Serum Albumin.

Amyloid-β (Aβ), Aβ40, Aβ42, and, recently, Aβ25-35 have been directly implicated in the pathogenesis of Alzheimer’s disease. We have studied the effects of Aβ on neuronal death, reactive oxygen species (ROS) production, and synaptic assembling in neurons in primary culture. Aβ25-35, Aβ40, and Aβ42 significantly decreased neuronal viability, although Aβ25-35 showed a higher effect. Aβ25-35 showed a more penetrating ability to reach mitochondria while Aβ40 did not enter the neuronal cytosol and Aβ42 was scarcely internalized. We did not observe a direct correlation between ROS production and cell death because both Aβ40 and Aβ42 decreased neuronal viability but Aβ40 did not change ROS production. Rather, ROS production seems to correlate with the penetrating ability of each Aβ. No significant differences were found between Aβ40 and Aβ42 regarding the extent of the deleterious effects of both peptides on neuronal viability or synaptophysin expression. However, Aβ40 elicited a clear delocalization of PSD-95 and synaptotagmin from prospective synapsis to the neuronal soma, suggesting the occurrence of a crucial effect of Aβ40 on synaptic disassembling. The formation of Aβ40- or Aβ42-serum albumin complexes avoided the effects of these peptides on neuronal viability, synaptophysin expression, and PSD-95/synaptotagmin disarrangement suggesting that sequestration of Aβ by albumin prevents deleterious effects of these peptides. We can conclude that Aβ borne by albumin can be safely transported through body fluids, a fact that may be compulsory for Aβ disposal by peripheral tissues.

Pax2+ astrocytes in the fish optic nerve head after optic nerve crush.

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2(+) astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2(+) astrocytes in this region. These Pax2(+) astrocytes show a higher number of Cytokeratin (Ck)(+)/GFAP(+) processes compared with control animals. In contrast, a different S100(+) astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2(+)/PCNA(+) that is activated after the injury. We conclude that Pax2(+) astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2(+) astrocytes are probably directly involved in the process of axonal regeneration.

Restrained Phosphatidylcholine Synthesis in a Cellular Model of Down’s Syndrome is Associated with the Overexpression of Dyrk1A

Aberrant formation of the cerebral cortex could be attributed to the lack of suitable substrates that direct the migration of neurons. Previous work carried out at our laboratory has shown that oleic acid is a neurotrophic factor. In order to characterize the effect of oleic acid in a cellular model of Down’s syndrome (DS), here, we used immortalized cell lines derived from the cortex of trisomy Ts16 and euploid mice. We report that in the plasma membrane of euploid cells, an increase in phosphatidylcholine concentrations occurs in the presence of oleic acid. However, in trisomic cells, oleic acid failed to increase phosphatidylcholine incorporation into the plasma membrane. Gene expression analysis of trisomic cells revealed that the phosphatidylcholine biosynthetic pathway was deregulated. Taken together, these results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21. DYRK1A contributes to intellectual disability and the early onset of Alzheimer’s disease in DS patients. Here, we explored the potential role of Dyrk1A in the reduction of phosphatidylcholine concentrations in trisomic cells in the presence of oleic acid. The downregulation of Dyrk1A by small interfering RNA (siRNA) in trisomic cells returned phosphatidylcholine concentrations up to similar levels to those of euploid cells in the presence of oleic acid. Thus, our results highlight the role of Dyrk1A in brain development through the modulation of phosphatidylcholine location, levels and synthesis.

Oleic Acid and Cholinergic dysfunction in Down Syndrome Models of the Central Nervous System

Down syndrome (DS): or trisomy 21: is the most common autosomal aneuploidy and the leading genetic cause of intellectual disability. It is widely established that mental retardation is primarily a consequence of brain functioning and developmental abnormalities in neurogenesis. Some changes in the physical structure of the dendrites are a major cause of impaired synaptic plasticity of DS. The overexpression of the dual specificyty tyrsone phosphorylation-regulated kinase 1A (DYRK1A): located on chromosome 21: is involved in cellular plasticity and responsible for central nervous system disturbance in DS. Oleic acid is a neurotrophic factor that promotes neuronal differentiation and increases the levels of choline acetyltransferase (ChAT). Furthermore: it has recently been shown that it induces migration and formation of new synapses in euploid cells. However: remarkably oleic acid fails to reproduce the same effects in trisomic cells. Here we review the hypothesis that oleic acid-dependent synaptic plasticity may be dependent on the lipid environment. Thus: differences in membrane composition may be essential to understand why oleic acid promotes higher cell plasticity in euploid than in trisomic cells.