Selective regulation of axonal growth from developing hippocampal neurons by tumor necrosis factor superfamily member APRIL.

APRIL (A Proliferation-Inducing Ligand, TNFSF13) is a member of the tumor necrosis factor superfamily that regulates lymphocyte survival and activation and has been implicated in tumorigenesis and autoimmune diseases. Here we report the expression and first known activity of APRIL in the nervous system. APRIL and one of its receptors, BCMA (B-Cell Maturation Antigen, TNFRSF17), are expressed by hippocampal pyramidal cells of fetal and postnatal mice. In culture, these neurons secreted APRIL, and function-blocking antibodies to either APRIL or BCMA reduced axonal elongation. Recombinant APRIL enhanced axonal elongation, but did not influence dendrite elongation. The effect of APRIL on axon elongation was inhibited by anti-BCMA and the expression of a signaling-defective BCMA mutant in these neurons, suggesting that the axon growth-promoting effect of APRIL is mediated by BCMA. APRIL promoted phosphorylation and activation of ERK1, ERK2 and Akt and serine phosphorylation and inactivation of GSK-3ß in cultured hippocampal pyramidal cells. Inhibition of MEK1/MEK2 (activators of ERK1/ERK2), PI3-kinase (activator of Akt) or Akt inhibited the axon growth-promoting action of APRIL, as did pharmacological activation of GSK-3ß and the expression of a constitutively active form of GSK-3ß. These findings suggest that APRIL promotes axon elongation by a mechanism that depends both on ERK signaling and PI3-kinase/Akt/GSK-3ß signaling.

A Uniform and Isotropic Cytoskeletal Tiling Fills Dendritic Spines.

Dendritic spines are submicron, subcellular compartments whose shape is defined by actin filaments and associated proteins. Accurately mapping the cytoskeleton is a challenge, given the small size of its components. It remains unclear whether the actin-associated structures analyzed in dendritic spines of neurons in vitro apply to dendritic spines of intact, mature neurons in situ. Here, we combined advanced preparative methods with multitilt serial section electron microscopy (EM) tomography and computational analysis to reveal the full three-dimensional (3D) internal architecture of spines in the intact brains of male mice at nanometer resolution. We compared hippocampal (CA1) pyramidal cells and cerebellar Purkinje cells in terms of the length distribution and connectivity of filaments, their branching-angles and absolute orientations, and the elementary loops formed by the network. Despite differences in shape and size across spines and between spine heads and necks, the internal organization was remarkably similar in both neuron types and largely homogeneous throughout the spine volume. In the tortuous mesh of highly branched and interconnected filaments, branches exhibited no preferred orientation except in the immediate vicinity of the cell membrane. We found that new filaments preferentially split off from the convex side of a bending filament, consistent with the behavior of Arp2/3-mediated branching of actin under mechanical deformation. Based on the quantitative analysis, the spine cytoskeleton is likely subject to considerable mechanical force in situ.

The Appearance Order of Varying Intervals Introduces Extra Modulation Effects on Neuronal Firing Through Non-linear Dynamics of Sodium Channels During High-Frequency Stimulations.

Electrical pulse stimulation in the brain has shown success in treating several brain disorders with constant pulse frequency or constant inter-pulse interval (IPI). Varying IPI may offer a variety of novel stimulation paradigms and may extend the clinical applications. However, a lack of understanding of neuronal responses to varying IPI limits its informed applications. In this study, to investigate the effects of varying IPI, we performed both rat experiments and computational modeling by applying high-frequency stimulation (HFS) to efferent axon fibers of hippocampal pyramidal cells. Antidromically evoked population spikes (PSs) were used to evaluate the neuronal responses to pulse stimulations with different IPI patterns including constant IPI, gradually varying IPI, and randomly varying IPI. All the varying IPI sequences were uniformly distributed in the same interval range of 10 to 5 ms (i.e., 100 to 200 Hz). The experimental results showed that the mean correlation coefficient of PS amplitudes to the lengths of preceding IPI during HFS with random IPI (0.72 ± 0.04, n = 7 rats) was significantly smaller than the corresponding correlation coefficient during HFS with gradual IPI (0.92 ± 0.03, n = 7 rats, P < 0.001, t-test). The PS amplitudes induced by the random IPI covered a wider range, over twice as much as that induced by the gradual IPI, indicating additional effects induced by merely changing the appearance order of IPI. The computational modeling reproduced these experimental results and provided insights into these modulatory effects through the mechanism of non-linear dynamics of sodium channels and potassium accumulation in the narrow peri-axonal space. The simulation results showed that the HFS-induced increase of extracellular potassium ([K+] o ) elevated the membrane potential of axons, delayed the recovery course of sodium channels that were repeatedly activated and inactivated during HFS, and resulted in intermittent neuronal firing. Because of non-linear membrane dynamics, random IPI recruited more neurons to fire together following specific sub-sequences of pulses than gradual IPI, thereby widening the range of PS amplitudes. In conclusion, the study demonstrated novel HFS effects of neuronal modulation induced by merely changing the appearance order of the same group of IPI of pulses, which may inform the development of new stimulation patterns to meet different demands for treating various brain diseases.

Differences in Reperfusion-Induced Mitochondrial Oxidative Stress and Cell Death Between Hippocampal CA1 and CA3 Subfields Are Due to the Mitochondrial Thioredoxin System.

AIMS: The susceptibility of CA1 over CA3 to damage from cerebral ischemia may be related to the differences in reactive oxygen species (ROS) production/removal between the two hippocampal subfields. We aimed to measure CA1/CA3 differences in net ROS production in real time in the first 30 min of reperfusion in pyramidal cells. We aimed to determine the underlying cause of the differential vulnerability of CA1 and CA3. RESULTS: Real-time determinations of mitochondrial H2O2 and, independently, glutathione (GSH) redox status from roGFP-based probes in individual pyramidal cells in organotypic hippocampal cultures during oxygen-glucose deprivation (OGD)-reperfusion (RP) demonstrate a significantly more oxidizing environment during RP in CA1 than CA3 mitochondria. Protein levels (immunohistochemistry and Western blots), roGFP2-based probe measurements during controlled mitochondrial production of ROS, and thioredoxin reductase (TrxR) inhibition by auranofin are consistent with a more effective mitochondrial thioredoxin (Trx) system in CA3. Inhibition of TrxR eliminates the differences in redox status and cell death between the regions. Overexpression of cytosolic Trx1 does not influence mitochondrial H2O2 production. INNOVATION: Real-time changes of mitochondrial H2O2 and GSH in tissue cultures during early RP, and also during controlled production of superoxide and peroxide, reveal significant differences between CA1 and CA3. The mitochondrial Trx system is responsible for the observed differences during RP as well as for delayed cell death 18 h afterward. CONCLUSION: Greater mitochondrial Trx efficacy in CA3 pyramidal cells results in less vulnerability to ischemia/reperfusion because of the less oxidizing environment in CA3 mitochondria during RP. Antioxid. Redox Signal. 27, 534-549.

A novel cell migratory zone in the developing hippocampal formation.

The hippocampal formation (HF) is a unique structure in the mammalian brain and is subdivided into the dentate gyrus, Ammon's horn, and subiculum by their functions and connectivity in the neuronal circuit. Because behaviors of the neural stem cells, neuronal progenitors, and the differentiating neurons are complex during hippocampal morphogenesis, the differentiation of these subdivisions has not been well understood. In this study, we investigated embryonic and postnatal expression of the proteins Prox1, Math2, and Ctip2, which clearly indicate principal cells of the dentate gyrus (Prox1 positive) and Ammon's horn (Math2 and Ctip2 positive). Expression patterns of Prox1 and Math2 were consistent with previously suggested localization of migratory pathways of the dentate granule cells and hippocampal pyramidal cells. Interestingly, we found intermingling of Prox1-expressing cells and Math2-expressing cells in a cell migratory stream, suggesting previously unknown behaviors of differentiating cells of the HF.