Jun Nishiyama

Aberrant Membranes and Double-Membrane Structures Accumulate in the Axons of Atg5-Null Purkinje Cells before Neuronal Death

Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes (Atg) have been identified in yeast; a ubiquitin-like Atg12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5flox/flox with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell–specific promoter, pcp2. In Atg5flox/flox; pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons.

Biochemical Computation for Spine Structural Plasticity

The structural plasticity of dendritic spines is considered to be essential for various forms of synaptic plasticity, learning, and memory. The process is mediated by a complex signaling network consisting of numerous species of molecules. Furthermore, the spatiotemporal dynamics of the biochemical signaling are regulated in a complicated manner because of geometrical restrictions from the unique morphology of the dendritic branches and spines. Recent advances in optical techniques have enabled the exploration of the spatiotemporal aspects of the signal regulations in spines and dendrites and have provided many insights into the principle of the biochemical computation that underlies spine structural plasticity.

Dynein- and activity-dependent retrograde transport of autophagosomes in neuronal axons

The accumulation of autophagosomes within axons is often observed in axonopathies associated with various neurological disorders, including those following excitotoxic insults. Nevertheless, the life cycle of autophagosomes in axons is not well understood. In the present study, we used microexplant cultures of cerebellar granule cells from GFP-LC3 transgenic mice to perform time-lapse imaging of LC3-positive dots in identified axons. Since these GFP-LC3 dots were never observed in granule cells on an Atg5-null background, they were considered to represent autophagosomes. Under physiological conditions, the autophagosomes showed bidirectional and saltatory movement with a bias towards one direction. Such vectorial movement was largely blocked by the dynein motor inhibitor EHNA (erythro-9-[3-(2-hydroxynonyl)] adenine), suggesting that the autophagosomes moved towards the soma, where most lysosomes are located. Interestingly, the application of the glutamate analog Ν-methyl-D-aspartic acid (NMDA) as an excitotoxin increased the number of autophagosomes in axons, while it did not significantly change its movement characteristics. These results suggest that autophagosomes play important roles in axons and are dynamically regulated under physiological and pathological conditions.

Cerebellar long-term depression requires dephosphorylation of TARP in Purkinje cells.

Cerebellar long‐term depression (LTD) at parallel fiber (PF)–Purkinje cell synapses is thought to play an essential role in certain forms of motor learning. Like hippocampal LTD, cerebellar LTD is mediated by the endocytosis of AMPA (α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate) receptors at postsynaptic sites. However, similar sets of kinases and phosphatases have opposite regulatory effects on hippocampal and cerebellar LTD, although the mechanisms responsible for this difference remain largely unclear. Activity‐dependent dephosphorylation of stargazin (an AMPA receptor auxiliary protein) by calcineurin regulates hippocampal LTD, but whether and how stargazin is involved in cerebellar LTD is unknown. In this study, we showed that stargazin is highly phosphorylated at basal states and is dephosphorylated by the application of high KCl plus glutamate (K‐glu) or of a metabotropic glutamate receptor agonist, (S)‐3,5‐dihydroxyphenylglycine (DHPG), both of which chemically induced LTD in cerebellar slices. This chemically induced dephosphorylation of stargazin was specifically blocked by a calcineurin inhibitor. Indeed, inclusion of the calcineurin auto‐inhibitory peptide in the patch pipette solution completely inhibited the LTD induced by the conjunctive stimulation of PFs and Purkinje cells. Furthermore, in Purkinje cells expressing stargazin‐9D, in which all nine serine residues are mutated to aspartate, neither conjunctive stimulus nor DHPG treatment induced LTD. Finally, immunohistochemical analyses revealed that neither K‐glu nor DHPG induced the endocytosis of AMPA receptors in Purkinje cells expressing stargazin‐9D. Together, these results indicate that hippocampal and cerebellar LTD share a common pathway, namely dephosphorylation of stargazin by calcineurin.

Selective and regulated gene expression in murine Purkinje cells by in utero electroporation

Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25–28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid‐related orphan receptor α (RORα1), were recapitulated by electroporating a dominant‐negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells’ physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells.

Reevaluation of Neurodegeneration in lurcher Mice: Constitutive Ion Fluxes Cause Cell Death with, Not by, Autophagy

The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding δ2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2Lc) triggers cell death has remained elusive. Here, taking advantage of recent knowledge about the domain structure of GluD2, we reinvestigated Lc-mediated cell death, focusing on the “autophagic cell death” hypothesis. Although autophagy and cell death were induced by the expression of GluD2Lc in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2Lc or by removal of extracellular Na+. In addition, although GluD2Lc is reported to directly activate autophagy, mutant channels that are not associated with n-PIST (neuronal isoform of protein-interacting specifically with TC10)–Beclin1 still caused autophagy and cell death. Furthermore, cells expressing GluD2Lc showed decreased ATP levels and increased AMP-activated protein kinase (AMPK) activities in a manner dependent on extracellular Na+. Thus, constitutive currents were likely necessary and sufficient to induce autophagy via AMPK activation, regardless of the n-PIST–Beclin1 pathway in vitro. Interestingly, the expression of dominant-negative AMPK suppressed GluD2Lc-induced autophagy but did not prevent cell death in heterologous cells. Similarly, the disruption of Atg5, a gene crucial for autophagy, did not prevent but rather aggravated Purkinje-cell death in Lc mice. Furthermore, calpains were specifically activated in Lc Purkinje cells. Together, these results suggest that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro.

A mechanism for congestion control based on traffic measurement with OpenFlow in disaster

Emergency Rescue Information Sharing System gathers information about victims and provides it. In disaster, usable bandwidth is not enough and there are many requests for communication. If information about victims is continuously transmitted to a link whose bandwidth is compressed, congestion of the link occurs, causing delay or loss of information. Therefore, in order to prevent information loss and delay, transmitting information in consideration of the bandwidth of wireless links is important. In this poster, we propose a congestion control method by adjusting transmission speed of information on victims and load balancing to each wireless link.

Genome editing in the mammalian brain using the CRISPR–Cas system

Recent advances in genome editing technologies such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 have enabled the rapid and efficient modification of endogenous genomes in a variety of cell types, accelerating biomedical research. In particular, precise genome editing in somatic cells in vivo allows for the rapid generation of genetically modified cells in living animals and holds great promise for the possibility of directly correcting genetic defects associated with human diseases. However, because of the limited efficiency and suitability of these technologies in the brain, especially in postmitotic neurons, the practical application of genome editing technologies has been largely limited in the field of neuroscience. Recent technological advances overcome significant challenges facing genome editing in the brain and have enabled us to precisely edit the genome in both mitotic cells and mature postmitotic neurons in vitro and in vivo, providing powerful means for studying gene function and dysfunction in the brain. This review highlights the development of genome editing technologies for the brain and discusses their applications, limitations, and future challenges.

An Efficient Image Gathering Scheme with Quality Control in Disaster

Rescue teams require information about victims in need of help rapidly to improve survival rate at the time of disaster. Emergency Rescue Information Sharing System gathers and provides the information with a framework called METHANE in addition to efficient wireless links selection using OpenFlow. However, it is difficult for those who rescue the victims except for the rescue teams to recognize detail situation of disaster area by using only METHANE information. To gather and provide the information rapidly, we introduce information about disaster area such as image information in addition to METHANE framework. However, transmission of the image information affects transmission of METHANE information in unstable network at the time of disaster. In this paper, we propose a transmission scheme using quality modification of image information. This scheme aims to gather high quality image information rapidly without affecting the transmission of METHANE information. To enable the transmission of the image information without affecting the transmission of METHANE, we distinguish flows of contents and assign different wireless links to each content. Moreover, for rapid image information gathering, we select the links dynamically based on available bandwidth and modify quality of information. Prototype demonstration shows that proposed scheme has usefulness in terms of rapid collection of low quality image information as well as slow collection of high quality image information.