Hiroyuki Yaginuma

Peptide inhibitors of the ice protease family arrest programmed cell death of motoneurons in vivo and in vitro

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

An anatomic study of the lumbar plexus with respect to retroperitoneal endoscopic surgery.

Study Design. The distribution of the lumbar plexus was analyzed using cadavers. Objective. To clarify the safety zone to prevent nerve injuries with respect to retroperitoneal endoscopic surgery. Summary of Background Data. Surgical approaches to the retroperitoneal space vary among surgeons. Recently, retroperitoneal endoscopic surgery has been applied to various spinal disorders. When the psoas major muscle is separated during retroperitoneal endoscopic surgery, there is a potential risk of injury to the lumbar plexus or nerve roots. However, there is sparse knowledge regarding the relationship between the greater psoas muscle and the lumbar plexus. Methods. A total of 30 cadavers were analyzed. Six lumbar spines of the cadavers were cut in parallel with the lumbar disc space. Each axial section was photographed and captured into a computer. The distribution of the lumbar plexus was analyzed using computer images. The positions where the genitofemoral nerve emerged on the abdominal surface of the psoas major muscle were analyzed using 24 cadavers. Results. L2/3 and above, all parts of the lumbar plexus, and nerve roots were located from the dorsal fourth of the vertebral body and dorsally. The genitofemoral nerve descends obliquely forward through the psoas major muscle, emerging on the abdominal surface between the cranial third of the L3 vertebra and the caudal third of the L4 vertebra. The safety zone of the psoas major muscle to prevent nerve injuries, excluding the genitofemoral nerve, is at L4/L5 and above. Conclusions. The safety zone, excluding the genitofemoral nerve, is at L4-L5 and above.

A Lentiviral Strategy for Highly Efficient Retrograde Gene Transfer by Pseudotyping with Fusion Envelope Glycoprotein

The lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) is used extensively in gene therapy trials of neurological and neurodegenerative diseases. Retrograde axonal transport of viral vectors offers a great advantage to the delivery of genes into neuronal cell bodies that are situated in regions distant from the injection site. Pseudotyping of HIV-1-based vectors with selective variants of rabies virus glycoprotein (RV-G) increases gene transfer via retrograde transport into the central nervous system. Because large-scale application for gene therapy trials requires high titer stocks of the vector, pseudotyping of a lentiviral vector that produces more efficient retrograde transport is needed. In the present study, we developed a novel vector system for highly efficient retrograde gene transfer by pseudotyping an HIV-1 vector with a fusion envelope glycoprotein (termed FuG-B) in which the cytoplasmic domain of RV-G was substituted by the corresponding part of vesicular stomatitis virus glycoprotein. The FuG-B pseudotype shifted the transducing property of the lentiviral vector and enhanced the retrograde transport-mediated gene transfer into different brain regions innervating the striatum with greater efficiency than that of the RV-G pseudotype in mice. In addition, injection of the FuG-B-pseudotyped vector into monkey striatum (caudate and putamen) allowed for highly efficient gene delivery into the nigrostriatal dopamine system, which is a major target for gene therapy of Parkinsons disease. Our strategy provides a powerful tool for the treatment of certain neurological and neurodegenerative diseases by promoting retrograde gene delivery via a lentiviral vector.

Survival of Developing Motor Neurons Mediated by Rho GTPase Signaling Pathway through Rho-Kinase

A variety of neurons generated during embryonic development survive or undergo programmed cell death (PCD) at later developmental stages. Survival or death of developing neurons is generally considered to depend on trophic support from various target tissues. The small GTPase Rho regulates diverse cellular processes such as cell morphology, cell adhesion, cell motility, and apoptosis. Rho-dependent serine–threonine protein kinase (Rho-kinase–ROK–ROCK), one of the effector proteins, transmits signals for some Rho-mediated processes. Here, we report the in vivo role of the Rho signaling pathway through Rho-kinase during development of motor neurons (MNs) in the spinal cord. We performed conditional expression of a dominant-negative form for RhoA (RhoA DN) or for Rho-kinase (Rho-K DN) in transgenic mice by using the Cre-loxP system to suppress the activity of these signaling molecules in developing MNs. Expression of RhoA DN reduced the number of MNs in the spinal cord because of increased apoptosis while preserving the gross patterning of motor axons. Expression of Rho-K DN produced developmental defects similar to those observed in RhoA DN expression. In addition, analysis of transgenic mice expressing Rho-K DN showed that the increased apoptosis of MNs was induced at the early embryonic stages before the initiation of PCD, and that MN death at the late embryonic stages corresponding to the period of PCD was moderately enhanced in the transgenic mice. These findings indicate that the Rho signaling pathway, primarily through Rho-kinase, plays a crucial role in survival of spinal MNs during embryogenesis, particularly at the early developmental stages.

Spinocerebellar projection fields in the horizontal plane of lobules of the cerebellar anterior lobe in the cat: an anterograde wheat germ agglutinin-horseradish peroxidase study

Using the anterograde transport of wheat germ agglutinin-horseradish peroxidase in the cat, the projection fields of the spinocerebellar tracts were studied in the horizontal plane of lobules of the anterior lobe. The central cervical nucleus projected to 3 longitudinal areas in zone A and the medial part of zone B. The T4-T7 segments projected to two major longitudinal areas in zones A and B, and small areas in zones C1-D. The L2-L4 segments projected densely to longitudinal areas in zone A and broad band-like areas in zones B-D. The L5 and L6 segments projected mainly to less demarcated areas in apical parts of zones B-D. The findings suggest that the basic pattern of projection proper to each spinocerebellar tract is consistent in all lobules of termination.

Expression pattern of LRR and Ig domain-containing protein (LRRIG protein) in the early mouse embryo.

The combination of leucine-rich repeat (LRR) and immunoglobulin-like (Ig) domains is found in the domain architecture of the Trk neurotrophin receptor protein. Recently dozens of such proteins simultaneously carrying LRR and Ig domains as the Trk receptors have been identified. Given the significant biological roles of Trk and such newly identified proteins, we have searched the public database for human proteins with LRR and Ig domains (collectively termed the leucine-rich repeat and Ig domain-containing protein, LRRIG protein, in this study), and have analyzed the mRNA expression pattern of mouse orthologs of obtained human LRRIG proteins at embryonic day 10. The list of the LRRIG proteins includes 36 human proteins: four LINGO, three NGL, five SALM, three NLRR, three Pal, two ISLR, three LRIG, two GPR, two Adlican, two Peroxidasin-like proteins, three Trk neurotrophin receptors, a yet unnamed protein AAI11068, and three AMIGO. Some molecules (LINGO2, LINGO4, NGL1, SALM1, SALM5, and TrkB) were expressed exclusively in neuronal tissues, whereas others (ISLR1, GPR124, and Adlican2) exhibited non-neuronal expression profiles. However, the majority of LRRIG protein family exhibited broad mRNA tissue-expression profiles.

Modulation of early but not later stages of programmed cell death in embryonic avian spinal cord by sonic hedgehog.

Sonic hedgehog (Shh) is a secreted glycoprotein expressed by the notochord and floor plate that is involved in the induction and specification of ventral phenotypes in the vertebrate neural tube. Recently, Shh has also been shown to promote the survival of cultured rat embryo ventral brain and spinal cord cells. We have examined whether Shh can promote the survival of chick embryo neurons in vivo or in vitro. In the chick, Shh is expressed in notochord, floor plate, and ventral neural tube/spinal cord at several stages at which programmed cell death (PCD) occurs. However, the administration of exogenous Shh to embryos in vivo or to motoneuron cultures at these stages failed to promote the survival of several different neuronal populations, including spinal motoneurons, spinal interneurons, sympathetic preganglionic neurons, sensory neurons, and neuronal precursor cells. Rather, at the earliest stage of PCD examined here (embryonic day 3) Shh selectively induced the death of ventral neuronal precursors and floor-plate cells, resulting in a net loss of cells in the neural tube. Altered concentrations of Shh induce aberrant phenotypes that are removed by PCD. Accordingly, normal PCD in the early neural tube may play a role in dorsal-ventral patterning.

Caspase activity is involved in, but is dispensable for, early motoneuron death in the chick embryo cervical spinal cord

We examined the role of caspases in the early programmed cell death (PCD) of motoneurons (MNs) in the chick embryo cervical cord between embryonic day (E) 4 and E5. An increase in caspase-3-like activity in MNs was observed at E4.5. Treatment with an inhibitor of caspase-3-like activity, Ac-DEVD-CHO, for 12 h blocked this increase and revealed that caspase-3-like activity is mainly responsible for DNA fragmentation and the nuclear changes during PCD but not for degenerative changes in the cytoplasm. When a more broad-spectrum caspase inhibitor was used (bocaspartyl (OMe)-fluoromethyl ketone, BAF), the appearance of degenerative changes in the cytoplasm was delayed by at least 12 h. However, following treatment with either Ac-DEVD-CHO or BAF for 24 h, the number of surviving healthy MNs did not differ from controls, indicating a normal occurrence of PCD despite the inhibition of caspases. These results suggest that caspase cascades that occur upstream of and are independent of the activation of caspase-3-like activity are responsible for the degenerative changes in the cytoplasm of dying cervical MNs. These data also suggest that, although one function of caspases may be to facilitate the kinetics of PCD, caspases are nonetheless dispensable for at least some forms of normal neuronal PCD in vivo.

Differential distribution of spinocerebellar fiber terminals within the lobules of the cerebellar anterior lobe in the cat: an anterograde WGA-HRP study.

The main termination field of spinocerebellar fibers in the anterior lobe was studied using anterograde transport of WGA-HRP in the cat. The C1 to C4 segments project to a midline region of the basal part of lobules I to V. The T3 and T4 or the T4 to T7 segments project mainly within a distance of 1.0 mm lateral to the midline in the middle part of lobules I to V whereas the L2 and L3 segments project mainly to a zone extending from 1.0 mm to 2.0 mm lateral to the midline in the middle part of these lobules. The L7 to Ca3 segments project to a zone extending from 1.0 mm to 2.0 mm lateral to the midline in the apical part of the lobules. The results indicate that the folium of the cerebellar anterior lobe is functionally differentiated from the medial to lateral part and from the apical to the basal part.

Identification and characterization of a novel member of murine semaphorin family

The semaphorin gene family contains a large number of secreted type or transmembrane type proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. Here we report a novel member of murine class 3 semaphorin genes, semaphorin 3G (Sema3G), mapped on chromosome 14. In adulthood, Sema3G is mainly expressed in the lung and kidney, and a little in the brain. Interestingly, in the adult rodent brain Sema3G is expressed only in the granular layer of the cerebellum, as determined by Northern blot and in situ hybridization analyses. We also found that Sema3G binds Neuropilin‐2, but not Neuropilin‐1, and induces the repulsion of sympathetic axons, but not dorsal root ganglion axons, indicating that Sema3G utilizes Neuropilin‐2 as a receptor to repel specific types of axons.