Naoki Kotake

Muscle regeneration occurs to coincide with mitochondrial biogenesis

We investigated the expression of the nuclear-encoded genes controlling the mitochondrial properties in the mouse gastrocnemius muscle to gain insight into the mitochondrial biogenesis that occurs during the muscle degeneration/regeneration induced by freezing. In addition, we tested whether the muscle regeneration is affected by pharmacologically blocking the mitochondrial protein synthesis to elucidate the possible involvement of mitochondrial biogenesis in muscle regeneration. The activity of citrate synthase dramatically increased soon after the initial injury when the myoblasts began to differentiate into myotubes, indicating that mitochondrial biogenesis occurs early during the muscle regeneration. At the same time, the expression of mitochondrial biogenesis-related genes including PGC-1β, PRC, NRF-1, NRF-2, TFAM, mtSSB, fission 1, and Lon protease synchronized with that of the myogenic regulatory genes including MyoD and myogenin. The skeletal muscles forced to regenerate in the presence of chloramphenicol to block the mitochondrial protein synthesis were of poor repair with small myofibers and an increased amount of connective tissue. These results suggest that mitochondrial biogenesis activated early during the muscle regeneration and that mitochondrial biogenesis plays a role in muscle regeneration.

Mitochondrial adaptations in skeletal muscle to hindlimb unloading

To gain insight into the regulation of mitochondrial adaptations to hindlimb unloading (HU), the activity of mitochondrial enzymes and the expression of nuclear-encoded genes which control mitochondrial properties in mouse gastrocnemius muscle were investigated. Biochemical and enzyme histochemical analysis showed that subsarcolemmal mitochondria were lost largely than intermyofibrillar mitochondria after HU. Gene expression analysis revealed disturbed or diminished gene expression patterns. The three main results of this analysis are as follows. First, in contrast to peroxisome proliferator-activated receptor γ coactivator 1 β (PGC-1β) and PGC-1-related coactivator, which were down-regulated by HU, PGC-1α was up-regulated concomitant with decreased expression of its DNA binding transcription factors, PPARα, and estrogen-related receptor α (ERRα). Moreover, there was no alteration in expression of nuclear respiratory factor 1, but its downstream target gene, mitochondrial transcription factor A, was down-regulated. Second, both mitofusin 2 and fission 1, which control mitochondrial morphology, were down-regulated. Third, ATP-dependent Lon protease, which participates in mitochondrial-protein degradation, was also down-regulated. These findings suggest that HU may induce uncoordinated expression of PGC-1 family coactivators and DNA binding transcription factors, resulting in reducing ability of mitochondrial biogenesis. Furthermore, down-regulation of mitochondrial morphology-related genes associated with HU may be also involved in alterations in intracellular mitochondrial distribution.

Formation of one-way-structured cultured neuronal networks in microfluidic devices combining with micropatterning techniques

We present a simple method to regulate the direction of axon development in cultured neurons using microfabrication and microfluidics techniques. We fabricate a PDMS-based device and place it onto a chemically micropatterned glass substrate. We confirm that cultured neurons extend neurites along the medium flow direction and the micropatterned regions.

Pharmacological inhibition of HSP90 activity negatively modulates myogenic differentiation and cell survival in C2C12 cells

Heat-shock protein90 (HSP90) plays an essential role in maintaining stability and activity of its clients. HSP90 is involved in cell differentiation and survival in a variety of cell types. To elucidate the possible role of HSP90 in myogenic differentiation and cell survival, we examined the time course of changes in the expression of myogenic regulatory factors, intracellular signaling molecules, and anti-/pro-apoptotic factors when C2C12 cells were cultured in differentiation condition in the presence of a HSP90-specific inhibitor, geldanamycin. Furthermore, we examined the effects of geldanamycin on muscle regeneration in vivo. Our results showed that geldanamycin inhibited myogenic differentiation with decreased expression of MyoD, myogenin and reduced phosphorylation levels of Akt1. Geldanamycin had little effect on the phosphorylation levels of p38MAPK and ERK1/2 but reduced the phosphorylation levels of JNK. Along with myogenic differentiation, geldanamycin increased apoptotic nuclei with decreased expression of Bcl-2. The skeletal muscles forced to regenerate in the presence of geldanamycin were of poor repair with small regenerating myofibers and increased connective tissues. Together, our findings suggest that HSP90 may modulate myogenic differentiation and may be involved in cell survival.

Spatial and temporal expression of hypoxia-inducible factor-1α during myogenesis in vivo and in vitro

We investigated the spatial and temporal expression patterns of hypoxia-inducible factor-1α (HIF-1α) during muscle regeneration and myogenesis in a C2C12 cell culture system. The expression of HIF-1α synchronized with that of myogenic regulatory genes during muscle regeneration at both the mRNA and protein levels. The HIF-1α protein was localized in the nuclei of newly formed regenerating myofibers in three different muscle injury models, including freezing, bupivacaine injection, and muscular dystrophy. In myogenic cell culture, the HIF-1α protein was localized in the nucleus and cytoplasm of the majority of myoblasts and myotubes. HIF-1α protein expression decreased concomitant with the increased expression of MyoD and myogenin proteins after the induction of myogenic differentiation. We investigated the adaptive response of myoblasts to hypoxia-like conditions induced by treatment of cobalt chloride. This treatment allowed HIF-1α to accumulate and translocate to the nucleus to activate transcription of its target genes, suggesting that myoblasts adapted to acute hypoxia-like conditions through enhancing an HIF-1-dependent pathway. Our results provide insight into the possible involvement of HIF-1α in myogenesis in vivo and in vitro.

Bundled Microfluidic Channels for Nerve Regeneration Electrodes

The development of a neural interface that allows signals from the human nervous system to control external equipment is extremely important for the next generation of prosthetic systems. A novel multichannel regeneration-type nerve electrode that was designed to record from and stimulate peripheral nerves has been developed to allow for the control of artificial hands and to generate artificial sensations. In this study we propose a neural probe with multiple microfluidic channels for measuring signals from regenerated axons passing through the fluidic channels. Each channel also serves as a guidance tube of the nerve regeneration and as fluidic pathways for injecting chemicals such as nerve growth factors (NGF). Parylene microfluidic channels were formed with microelectrodes and then the substrate was rolled up, resulting in bundled channels. We have succeeded in injecting NGF from the channels and observed the regeneration of an axon guided by the channels.

Microfabrication- and microfluidics-based patterning of cultured neuronal network

The cultured neuronal monolayer has been a promising model system for studying the neuronal dynamics, from single cell to network-wide level. Randomness in the reconstituted network structure has, however, hindered regulated signal transmissions from one neuron to another or from one neuronal population to another. Applying microfabrication-based cell patterning techniques is a promising approach to handling these problems. In the present study, we attempt to regulate the direction of axon development and the pathway of signal transmissions in cultured neuronal networks using micro-fabrication and — fluidic techniques. We created a PDMS-based culture device, which consisted of arrays of U-shaped cell trapping microwells, and placed it onto a chemically micropatterned glass substrate. After 6 days in vitro, we confirmed that cortical neurons extended neurites along the medium flow direction and the micropatterned regions.

A Flexible Regeneration Microelectrode with Cell-Growth Guidance

In this study we fabricate a cell-guide on a thin disc-shaped probe with numerous microholes to be implanted between the severed stumps of axons; the cell-guide directs the regenerating axons though the holes in our probe (Fig. 1(a)). Since cell growth is greatly influenced by the topography of the substrate [1-3] we design a SU8 microfluidic cell-guide with a particular shape (Fig. 1(b)) to prevent the random spreading of cells by directing cell growth through the holes. This guide may reduce the chance of short-circuits of the probe by cells that span two different electrodes. Furthermore, as the cells are directed to grow through the holes, we can measure their electrical potential with gold electrodes that we micro-fabricate.

Heterogeneous Redistribution of Facial Subcategory Information Within and Outside the Face-Selective Domain in Primate Inferior Temporal Cortex

Abstract The inferior temporal cortex (ITC) contains neurons selective to multiple levels of visual categories. However, the mechanisms by which these neurons collectively construct hierarchical category percepts remain unclear. By comparing decoding accuracy with simultaneously acquired electrocorticogram (ECoG), local field potentials (LFPs), and multi-unit activity in the macaque ITC, we show that low-frequency LFPs/ECoG in the early evoked visual response phase contain sufficient coarse category (e.g., face) information, which is homogeneous and enhanced by spatial summation of up to several millimeters. Late-induced high-frequency LFPs additionally carry spike-coupled finer category (e.g., species, view, and identity of the face) information, which is heterogeneous and reduced by spatial summation. Face-encoding neural activity forms a cluster in similar cortical locations regardless of whether it is defined by early evoked low-frequency signals or late-induced high-gamma signals. By contrast, facial subcategory-encoding activity is distributed, not confined to the face cluster, and dynamically increases its heterogeneity from the early evoked to late-induced phases. These findings support a view that, in contrast to the homogeneous and static coarse category-encoding neural cluster, finer category-encoding clusters are heterogeneously distributed even outside their parent category cluster and dynamically increase heterogeneity along with the local cortical processing in the ITC.

A flexible parylene-based glutamate sensor

This paper presents the results of a study aimed at the fabrication and basic evaluation of a parylene-based flexible sensor for glutamate detection in the brain. We fabricated a flexible sensor using Micro Electro Mechanical Systems (MEMS) technology. Then, to modify the electrodes, we deposited an enzyme on the surface of the electrodes. To evaluate the function of the sensor, we performed an in vitro aliquot injection test. The fabricated flexible sensor was able to detect glutamate in vitro.