Tangential migration of neuronal precursors of glutamatergic neurons in the adult mammalian brain.

In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial "whole-mount" dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. VIDEO ABSTRACT.

Neuronal activity controls the antagonistic between PGC-1α and SMRT in regulating antioxidant defences

Transcriptional coactivators and corepressors often have multiple targets and can have opposing actions on transcription and downstream physiological events. The coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is under-expressed in Huntington's disease and is a regulator of antioxidant defenses and mitochondrial biogenesis. We show that in primary cortical neurons, expression of PGC-1α strongly promotes resistance to excitotoxic and oxidative stress in a cell autonomous manner, whereas knockdown increases sensitivity. In contrast, the transcriptional corepressor silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) specifically antagonizes PGC-1α-mediated antioxidant effects. The antagonistic balance between PGC-1α and SMRT is upset in favor of PGC-1α by synaptic activity. Synaptic activity triggers nuclear export of SMRT reliant on multiple regions of the protein. Concommitantly, synaptic activity post-translationally enhances the transactivating potential of PGC-1α in a p38-dependent manner, as well as upregulating cyclic-AMP response element binding protein-dependent PGC-1α transcription. Activity-dependent targeting of PGC-1α results in enhanced gene expression mediated by the thyroid hormone receptor, a prototypical transcription factor coactivated by PGC-1α and repressed by SMRT. As a consequence of these events, SMRT is unable to antagonize PGC-1α-mediated resistance to oxidative stress in synaptically active neurons. Thus, PGC-1α and SMRT are antagonistic regulators of neuronal vulnerability to oxidative stress. Further, this coactivatorcorepressor antagonism is regulated by the activity status of the cell, with implications for neuronal viability.

Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury.

Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.

Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF(-)GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF(-)GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in 'equivalent-light'' scenarios.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

Neuronal and endothelial nitric oxide synthase gene knockout mice

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

Effects of a neuronal nitric oxide synthase inhibitor on lipopolysaccharide-induced fever

It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg/kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02). The coinjection of LPS and 7-NI was followed by a significant (P<0.02) hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 ± 304; C to F1 (C x A) = 3899 ± 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 ± 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 ± 287; B to B = 2759 ± 170, and A to A = 2835 ± 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration.

Sulfated proteoglycans as modulators of neuronal migration and axonal decussation in the developing midbrain

Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions.

Functional role of a specific ganglioside in neuronal migration and neurite outgrowth

Cell migration occurs extensively during mammalian brain development and persists in a few regions in the adult brain. Defective migratory behavior of neurons is thought to be the underlying cause of several congenital disorders. Knowledge of the dynamics and molecular mechanisms of neuronal movement could expand our understanding of the normal development of the nervous system as well as help decipher the pathogenesis of neurological developmental disorders. In our studies we have identified and characterized a specific ganglioside (9-O-acetyl GD3) localized to the membrane of neurons and glial cells that is expressed in regions of cell migration and neurite outgrowth in the developing and adult rat nervous system. In the present article we review our findings that demonstrate the functional role of this molecule in neuronal motility.

Expression of neuronal nitric oxide synthase in the developing superficial layers of the rat superior colliculus

We investigated the level of expression of neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of the rat superior colliculus during early postnatal development. Male and female Lister rats ranging in age between the day of birth (P0) and the fourth postnatal week were used in the present study. Two biochemical methods were used, i.e., in vitro measurement of NOS specific activity by the conversion of [³H]-arginine to [³H]-citrulline, and analysis of Western blotting immunoreactive bands from superior colliculus homogenates. As revealed by Western blotting, very weak immunoreactive bands were observed as early as P0-2, and their intensity increased progressively at least until P21. The analysis of specific activity of NOS showed similar results. There was a progressive increase in enzymatic activity until near the end of the second postnatal week, and a nonsignificant tendency to an increase until the end of the third week was also observed. Thus, these results indicated an increase in the amount of nNOS during the first weeks after birth. Our results confirm and extend previous reports using histochemistry for NADPH-diaphorase and immunocytochemistry for nNOS, which showed a progressive increase in the number of stained cells in the superficial layers during the first two postnatal weeks, reaching an adult pattern at the end of the third week. Furthermore, our results suggested that nNOS is present in an active form in the rat superior colliculus during the period of refinement of the retinocollicular pathway.