MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

Blocking insulin-like growth factor 1 receptor signaling pathway inhibits neuromuscular junction regeneration after botulinum toxin-A treatment.

Botulinum toxin-A (BTX) administration into muscle is an established treatment for conditions with excessive muscle contraction. However, botulinum therapy has short-term effectiveness, and high-dose injection of BTX could induce neutralizing antibodies against BTX. Therefore, prolonging its effects could be beneficial in a clinical situation. Insulin-like growth factor-1 receptor (IGF1R) and its ligands, insulin-like growth factor (IGF) -I and II, regulate the physiological and pathological processes of the nervous system. It has been suggested that IGF1R is involved in the process after BTX administration, but the specific regeneration mechanism remains unclear. Therefore, this study aimed to determine how inhibition of IGF1R signaling pathway affects BTX-induced muscle paralysis. The results showed that anti-IGF1R antibody administration inhibited the recovery from BTX-induced neurogenic paralysis, and the synaptic components at the neuromuscular junction (NMJ), mainly post-synaptic components, were significantly affected by the antibody. In addition, the wet weight or frequency distribution of the cross-sectional area of the muscle fibers was regulated by IGF1R, and sequential antibody administration following BTX treatment increased the number of Pax7+-satellite cells in the gastrocnemius (GC) muscle, independent of NMJ recovery. Moreover, BTX treatment upregulated mammalian target of rapamycin (mTOR)/S6 kinase signaling pathway, HDAC4, Myog, Fbxo32/MAFbx/Atrogin-1 pathway, and transcription of synaptic components, but not autophagy. Finally, IGF1R inhibition affected only mTOR/S6 kinase translational signaling in the GC muscle. In conclusion, the IGF1R signaling pathway is critical for NMJ regeneration via specific translational signals. IGF1R inhibition could be highly beneficial in clinical practice by decreasing the number of injections and total dose of BTX due to the prolonged duration of the effect.

Influence of novel readthrough agents on myelin protein zero translation in the peripheral nervous system.

Translational readthrough-inducing agents have been developed for the treatment of nonsense mutations in hereditary diseases. The clinical effectiveness of readthrough agents has been reported, although newly developed agents are still desired because of their toxicities or limited clinical effectiveness. Recently, novel negamycin-derived readthrough agents without antimicrobial activity have been developed. Our aim was to evaluate the activities of these readthrough agents by monitoring the production of large myelin protein zero (L-MPZ), the programmed translational readthrough isoform of myelin protein zero (P0, MPZ) mRNA, and to clarify the influence of these agents on the sciatic nerve in vivo. First, we examined the readthrough activities of novel negamycin-derived agents using cell-free and cell culture systems using plasmids encoding human MPZ (hP0) cDNA. Three of the negamycin derivatives, TCP-112, TCP-169, and TCP-1109, suppressed the canonical stop codon to induce readthrough. Direct injection of TCP-1109, which showed higher readthrough activity for Mpz in mouse sciatic nerves, exhibited a 1.3-fold increase in the L-MPZ/P0 ratio compared to that with the vehicle control on western blotting. The nerve conduction velocity and beam walk test showed abnormalities in the classical readthrough agent G418-treated group, but not in the TCP-1109-treated group. Immunofluorescence analysis showed that TCP-1109 caused less damage to the sciatic nerve than G418. In the semi-thin sections, a lower g-ratio and more tomacula-like structures were observed in TCP-1109-treated nerves. Thus, the present results indicate that negamycin-derived readthrough agents enhance programmed translational readthrough, and the management of readthrough activities using canonical stop codons may be important.

Maternal and Adult Interleukin-17A Exposure and Autism Spectrum Disorder.

Epidemiological evidence in humans has suggested that maternal infections and maternal autoimmune diseases are involved in the pathogenesis of autism spectrum disorder. Animal studies supporting human results have shown that maternal immune activation causes brain and behavioral alterations in offspring. Several underlying mechanisms, including interleukin-17A imbalance, have been identified. Apart from the pro-inflammatory effects of interleukin-17A, there is also evidence to support the idea that it activates neuronal function and defines cognitive behavior. In this review, we examined the signaling pathways in both immunological and neurological contexts that may contribute to the improvement of autism spectrum disorder symptoms associated with maternal blocking of interleukin-17A and adult exposure to interleukin-17A. We first describe the epidemiology of maternal immune activation then focus on molecular signaling of the interleukin-17 family regarding its physiological and pathological roles in the embryonic and adult brain. In the future, it may be possible to use interleukin-17 antibodies to prevent autism spectrum disorder.

Do Neurotrophins Connect Neurological Disorders and Heart Diseases?

Neurotrophins (NTs) are one of the most characterized neurotrophic factor family members and consist of four members in mammals. Growing evidence suggests that there is a complex inter- and bi-directional relationship between central nervous system (CNS) disorders and cardiac dysfunction, so-called "brain-heart axis". Recent studies suggest that CNS disorders, including neurodegenerative diseases, stroke, and depression, affect cardiovascular function via various mechanisms, such as hypothalamic-pituitary-adrenal axis augmentation. Although this brain-heart axis has been well studied in humans and mice, the involvement of NT signaling in the axis has not been fully investigated. In the first half of this review, we emphasize the importance of NTs not only in the nervous system, but also in the cardiovascular system from the embryonic stage to the adult state. In the second half, we discuss the involvement of NTs in the pathogenesis of cardiovascular diseases, and then examine whether an alteration in NTs could serve as the mediator between neurological disorders and heart dysfunction. The further investigation we propose herein could contribute to finding direct evidence for the involvement of NTs in the axis and new treatment for cardiovascular diseases.

Pathophysiological Roles of Abnormal Axon Initial Segments in Neurodevelopmental Disorders.

The 20-60 µm axon initial segment (AIS) is proximally located at the interface between the axon and cell body. AIS has characteristic molecular and structural properties regulated by the crucial protein, ankyrin-G. The AIS contains a high density of Na+ channels relative to the cell body, which allows low thresholds for the initiation of action potential (AP). Molecular and physiological studies have shown that the AIS is also a key domain for the control of neuronal excitability by homeostatic mechanisms. The AIS has high plasticity in normal developmental processes and pathological activities, such as injury, neurodegeneration, and neurodevelopmental disorders (NDDs). In the first half of this review, we provide an overview of the molecular, structural, and ion-channel characteristics of AIS, AIS regulation through axo-axonic synapses, and axo-glial interactions. In the second half, to understand the relationship between NDDs and AIS, we discuss the activity-dependent plasticity of AIS, the human mutation of AIS regulatory genes, and the pathophysiological role of an abnormal AIS in NDD model animals and patients. We propose that the AIS may provide a potentially valuable structural biomarker in response to abnormal network activity in vivo as well as a new treatment concept at the neural circuit level.

Upregulation of large myelin protein zero leads to Charcot-Marie-Tooth disease-like neuropathy in mice.

Charcot-Marie-Tooth (CMT) disease is a hereditary neuropathy mainly caused by gene mutation of peripheral myelin proteins including myelin protein zero (P0, MPZ). Large myelin protein zero (L-MPZ) is an isoform of P0 that contains an extended polypeptide synthesized by translational readthrough at the C-terminus in tetrapods, including humans. The physiological role of L-MPZ and consequences of an altered L-MPZ/P0 ratio in peripheral myelin are not known. To clarify this, we used genome editing to generate a mouse line (L-MPZ mice) that produced L-MPZ instead of P0. Motor tests and electrophysiological, immunohistological, and electron microscopy analyses show that homozygous L-MPZ mice exhibit CMT-like phenotypes including thin and/or loose myelin, increased small-caliber axons, and disorganized axo-glial interactions. Heterozygous mice show a milder phenotype. These results highlight the importance of an appropriate L-MPZ/P0 ratio and show that aberrant readthrough of a myelin protein causes neuropathy.

Chronic peripheral nerve compression disrupts paranodal axoglial junctions.

INTRODUCTION: Peripheral nerves are often exposed to mechanical stress leading to compression neuropathies. The pathophysiology underlying nerve dysfunction by chronic compression is largely unknown. METHODS: We analyzed molecular organization and fine structures at and near nodes of Ranvier in a compression neuropathy model in which a silastic tube was placed around the mouse sciatic nerve. RESULTS: Immunofluorescence study showed that clusters of cell adhesion complex forming paranodal axoglial junctions were dispersed and overlapped frequently with juxtaparanodal components. These paranodal changes occurred without internodal myelin damage. The distribution and pattern of paranodal disruption suggests that these changes are the direct result of mechanical stress. Electron microscopy confirmed loss of paranodal axoglial junctions. CONCLUSIONS: Our data show that chronic nerve compression disrupts paranodal junctions and axonal domains required for proper peripheral nerve function. These results provide important clues toward better understanding of the pathophysiology underlying nerve dysfunction in compression neuropathies. Muscle Nerve 55: 544-554, 2017.

Microglial phospholipase D4 deficiency influences myelination during brain development.

Phospholipase D4 (PLD4) is expressed in activated microglia that transiently appear in white matter during postnatal brain development. Previous knockdown experiments using cultured microglia showed PLD4 involvement in phagocytosis and proliferation. To elucidate the role of PLD4 in vivo, PLD4-deficient mice were generated and the cerebella were examined at postnatal day 5 (P5) and P7, when PLD4 expression is highest in microglia. Wild type microglia showed strong immunoreactivity for microglial marker CD68 at P5, whereas CD68 signals were weak in PLD4-deficient microglia, suggesting that loss of PLD4 affects microglial activation. At P5 and P7, immunostaining for anti-myelin basic protein (MBP) antibody indicated a mild but significant delay in myelination in PLD4-deficient cerebellum. Similar change was also observed in the corpus callosum at P7. However, this difference was not apparent at P10, suggesting that microglial PLD4-deficiency primarily influences the early myelination stage. Thus, microglia may have a transient role in myelination via a PLD4-related mechanism during development.

Submembranous cytoskeletons stabilize nodes of Ranvier.

Rapid action potential propagation along myelinated axons requires voltage-gated Na(+) (Nav) channel clustering at nodes of Ranvier. At paranodes flanking nodes, myelinating glial cells interact with axons to form junctions. The regions next to the paranodes called juxtaparanodes are characterized by high concentrations of voltage-gated K(+) channels. Paranodal axoglial junctions function as barriers to restrict the position of these ion channels. These specialized domains along the myelinated nerve fiber are formed by multiple molecular mechanisms including interactions between extracellular matrix, cell adhesion molecules, and cytoskeletal scaffolds. This review highlights recent findings into the roles of submembranous cytoskeletal proteins in the stabilization of molecular complexes at and near nodes. Axonal ankyrin-spectrin complexes stabilize Nav channels at nodes. Axonal protein 4.1B-spectrin complexes contribute to paranode and juxtaparanode organization. Glial ankyrins enriched at paranodes facilitate node formation. Finally, disruption of spectrins or ankyrins by genetic mutations or proteolysis is involved in the pathophysiology of various neurological or psychiatric disorders.

PLD$ is involved in phagocytosis of microglia: expression and localization changes of PLD4 are correlated with activation state of microglia.

Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

BACKGROUND: Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)(4)-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.