Impact of a pyridazine derivative on tripartite synapse ultrastructure in hippocampus: a three-dimensional analysis

IntroductionWe previously discovered a pyridazine derivative compound series that can improve cognitive functions in mouse models of Alzheimer’s disease. One of the advanced compounds from this series, LDN/OSU-0215111-M3, was selected as the preclinical development candidate. This compound activates local protein translation at the perisynaptic astrocytic process (PAP) and enhances synaptic plasticity sequentially. While biochemical evidence supports the hypothesis that the compound enhances the structural plasticity of the tripartite synapse, its direct structural impact has not been investigated.MethodsVolume electron microscopy was used to study the hippocampal tripartite synapse three-dimensional structure in 3-month-old wild-type FVB/NJ mice after LDN/OSU-0215111-M3 treatment.ResultsLDN/OSU-0215111-M3 increased the size of tertiary apical dendrites, the volume of mushroom spines, the proportion of mushroom spines containing spine apparatus, and alterations in the spine distribution across the surface area of tertiary dendrites. Compound also increased the number of the PAP interacting with the mushroom spines as well as the size of the PAP in contact with the spines. Furthermore, proteomic analysis of the isolated synaptic terminals indicated an increase in dendritic and synaptic proteins as well as suggested a possible involvement of the phospholipase D signaling pathway. To further validate that LDN/OSU-0215111-M3 altered synaptic function, electrophysiological studies showed increased long-term potentiation following compound treatment.DiscussionThis study provides direct evidence that pyridazine derivatives enhance the structural and functional plasticity of the tripartite synapse

Myelination and axonal electrical activity modulate the distribution and motility of mitochondria at CNS nodes of Ranvier

Energy production presents a formidable challenge to axons as their mitochondria are synthesized and degraded in neuronal cell bodies. To meet the energy demands of nerve conduction, small mitochondria are transported to and enriched at mitochondrial stationary sites located throughout the axon. In this study, we investigated whether size and motility of mitochondria in small myelinated central nervous system axons was differentially regulated at nodes, and whether mitochondrial distribution and motility are modulated by axonal electrical activity. The size/volume of mitochondrial stationary sites was significantly larger in juxtaparanodal/internodal axoplasm than in nodal/paranodal axoplasm. By 3-dimensional electron microscopy, we observed that axonal mitochondrial stationary sites were composed of multiple mitochondria of varying length, except at nodes where mitochondria were uniformly short and frequently absent altogether. Mitochondrial transport speed was significantly reduced in nodal axoplasm when compared to internodal axoplasm. Increased axonal electrical activity decreased mitochondrial transport and increased the size of mitochondrial stationary sites in nodal/paranodal axoplasm. Decreased axonal electrical activity had the opposite effects. In cerebellar axons of the myelin deficient rat, which contains voltage-gated Na(+) channel clusters but lacks paranodal specializations, axonal mitochondrial motility and stationary site size were similar at Na(+) channel clusters and other axonal regions. These results demonstrate juxtaparanodal/internodal enrichment of stationary mitochondria and neuronal activity-dependent dynamic modulation of mitochondrial distribution and transport in nodal axoplasm. In addition, the modulation of mitochondrial distribution and motility requires oligodendrocyte-axon interactions at paranodal specializations

High Fat Diet-Induced Obesity Dysregulates Splenic B Cell Mitochondrial Activity

Diet-induced obesity impairs mitochondrial respiratory responses in tissues that are highly metabolically active, such as the heart. However, less is known about the impact of obesity on the respiratory activity of specific cell types, such as splenic B cells. B cells are of relevance, as they play functional roles in obesity-induced insulin resistance, inflammation, and responses to infection. Here, we tested the hypothesis that high-fat-diet (HFD)-induced obesity could impair the mitochondrial respiration of intact and permeabilized splenic CD19+ B cells isolated from C57BL/6J mice and activated ex vivo with lipopolysaccharide (LPS). High-resolution respirometry was used with intact and permeabilized cells. To reveal potential mechanistic targets by which HFD-induced obesity dysregulates B cell mitochondria, we conducted proteomic analyses and 3D serial block face scanning electron microscopy (SBFEM). High-resolution respirometry revealed that intact LPS-stimulated B cells of obese mice, relative to controls, displayed lower ATP-linked, as well as maximal uncoupled, respiration. To directly investigate mitochondrial function, we used permeabilized LPS-stimulated B cells, which displayed increased H2O2 emission and production with obesity. We also examined oxidative phosphorylation efficiency simultaneously, which revealed that oxygen consumption and ATP production were decreased in LPS-stimulated B cells with obesity relative to controls. Despite minimal changes in total respiratory complex abundance, in LPS-stimulated B cells of obese mice, three of the top ten most downregulated proteins were all accessory subunits of respiratory complex I. SBFEM showed that B cells of obese mice, compared to controls, underwent no change in mitochondrial cristae integrity but displayed increased mitochondrial volume that was linked to bioenergetic function. Collectively, these results establish a proof of concept that HFD-induced obesity dysregulates the mitochondrial bioenergetic metabolism of activated splenic B cells

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin, hnRNP A2 bound A2RE in the latter site with a K-d near 50 nM, whereas the K-d for hnRNP A1 was above 10 muM. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 muM for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis

Axons regulate the distribution of Schwann cell microtubules

It is well established that axons regulate Schwann cell phenotype. The purpose of the present study was to determine whether axons influence the arrangement of Schwann cell microtubules (MTs). Using double-labeling immunocytochemistry and confocal microscopy, we show that MTs in undifferentiated Schwann cells are nucleated from and attached to a single MT organizing center (MTOC) that is associated with the centrosome. Physical contact with appropriate axons initiates a myelin-forming phenotype that disperses MT minus ends and induces multiple MT-nucleating sites in Schwann cell perinuclear cytoplasm. The axonal signal that initiates myelin breakdown during Wallerian degeneration induces multiple MTOCs and MT bundles in Schwann cell perinuclear cytoplasm and in cytoplasm between degenerating myelin ovoids. These results establish that axons influence Schwann cell MT distribution by regulating the location and number of MT-nucleation sites