Asako Hase

Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle

Glial cell line–derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects on motor neurons as well as mesencephalic dopaminergic neurons. Because GDNF promotes survival of motor neurons in vivo and in vitro and rescues motor neurons from naturally occurring cell death, the potential use of GDNF for treatment of motor neuron diseases has been a major focus of recent research. The expression of GDNF in humans, however, has not been fully examined. In the present study, we examined the expression of GDNF in adult human muscle by Northern blot, reverse transcriptase polymerase chain reaction (RT‐PCR), and immunohistochemical analyses to address physiological roles of GDNF in humans. Northern blot analysis demonstrated high expression of GDNF mRNA in human skeletal muscle when compared to that of mouse. Intense GDNF immunoreactivity was observed in the vicinity of plasma membranes of skeletal muscle, particularly at neuromuscular junctions. GDNF immunoreactivity was also observed within the axons and surrounding Schwann cells of peripheral nerves. However, RT‐PCR detected expression of GDNF mRNA only in skeletal muscle, and not within the anterior horn cells of human spinal cord. These results suggest that GDNF is produced by skeletal muscle and taken up at the nerve terminals for retrograde transport by axons. Thus, GDNF in human skeletal muscle may be involved in promoting motor neuron survival as a target‐derived neurotrophic factor. J. Comp. Neurol. 402:303–312, 1998.

Expression of Dystroglycan and the Laminin-α2 Chain in the Rat Peripheral Nerve during Development

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan comprises the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha 2/beta 1/gamma 1), a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerves, we investigated the expression of beta-dystroglycan and the laminin-alpha 2 chain in rat sciatic nerve during development by immunoblot, immunofluorescence, and immunoelectron microscopic studies. The expression of beta-dystroglycan and the laminin-alpha 2 chain in the rat sciatic nerve was low and not confined to the Schwann cell outer membrane from embryonic day 18 to birth, when there was only an immature basal lamina assembly and no compact myelin formation by Schwann cells. However, the expression of these proteins increased markedly and became clearly localized to the Schwann cell outer membrane between birth and postnatal day 7, when both basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. From postnatal day 7 to adult, there was no remarkable change in the expression of these proteins. Our results support the hypothesis that the dystroglycan complex functions as an adhesion apparatus, binding the Schwann cell outer membrane with the basal lamina, and suggest that the dystroglycan complex plays a role in Schwann cell myelination through its interaction with laminin-2.

Characterization of glial cell line-derived neurotrophic factor family receptor α-1 in peripheral nerve Schwann cells

Glial cell line‐derived neurotrophic factor (GDNF) family receptor α‐1 (GFRα‐1) is a receptor component of GDNF that associates with and activates the tyrosine kinase receptor Ret. To further understand GDNF and its receptor system in the PNS, we first characterized the expression of GFRα‐1 in bovine peripheral nerve in vivo. GFRα‐1 immunoreactivity was localized adjacent to the outermost layer of myelin sheath, as well as in the endoneurium and axoplasm. In a fractionation study, GFRα‐1 was recovered mostly in the soluble fraction, although a small amount was recovered in the membrane fraction. A substantial amount of GFRα‐1 in the membrane fraction was extractable by detergent and alkaline conditions. To further clarify the expression of GFRα‐1 in Schwann cells, we examined cultured rat Schwann cells and the Schwannoma cell line RT4. Schwann cells expressed GFRα‐1 in both the soluble/cytosolic and membrane fractions, and the membrane form of GFRα‐1 was expressed at the outer surface of the Schwann cell plasma membrane. We also confirmed the secretion of the soluble form of GFRα‐1 from Schwannoma cells in a metabolic labeling experiment. These data contribute to our knowledge of the production, expression and functions of GFRα‐1 in the PNS.

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) expression in regenerating muscle fibers in neuromuscular diseases

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert a target-derived trophic factor for motor neurons. Immunohistochemical analyses revealed that expression of GDNF in regeneration muscle fibers was up-regulated in polymyositis (PM) and Duchenne type muscular dystrophy (DMD). Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the full length GDNF was up-regulated in PM and DMD muscle; normal muscle exhibited mostly truncated GDNF. The results indicate that the GDNF expression is regulated in regeneration of human skeletal muscle.

Expression of human GFRα-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFRα-1). Immunoreactivity of GFRα-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFRα-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFRα-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Expression of dystroglycan complex in satellite cells of dorsal root ganglia

Abstract. In Schwann cells, the transmembrane glycoprotein β-dystroglycan composes the dystroglycan complex together with the extracellular glycoprotein α-dystroglycan, which binds laminin-2 (α2/β1/γ1), a major component of the Schwann cell basal lamina. In the Schwann cell cytoplasm, β-dystroglycan is anchored to a dystrophin isoform, Dp116. In this study, we investigated the expression of β-dystroglycan, Dp116 and the laminin-α2 chain in satellite cells of rat dorsal root ganglia (DRGs). Immunohistochemical study showed that immunoreactivities for β-dystroglycan and Dp116 were both localized to the outer rim of neuron-satellite cell and axon-Schwann cell units, indicating that both satellite and Schwann cells expressed these proteins in DRGs. Immunoreactivity for the laminin-α2 chain was detected in a similar location, indicating that the basal lamina surrounding satellite and Schwann cells in DRGs contained laminin-2. Ultrastructurally, immunoreactivity for the cytoplasmic domain of β-dystroglycan as well as that for Dp116 was most intense in the cytoplasm just underlying the outer membrane of satellite cells. The immunoreactivity for laminin was associated with the outer surface of those cells, suggesting that it was localized in the surrounding basal lamina. These results indicate that the dystroglycan complex is expressed in the satellite cell outer membrane and involved in the adhesion with the basal lamina through the interaction with laminin-2.

Characterization of parkin in bovine peripheral nerve.

The autosomal recessive juvenile parkinsonism is caused by the mutations of the gene encoding a novel protein called parkin. It has been reported that parkin is expressed in the central nervous system and functions as a ubiquitin-protein ligase (E3) which suppresses neuronal cell degeneration by ubiquitinating misfolded proteins. Thus far, however, it remains unknown if parkin is expressed and functions in the peripheral nervous system. In order to begin to address to this question, we investigated the expression of parkin in bovine peripheral nerve. Reverse transcription polymerase chain reaction analysis demonstrated the presence of parkin transcript in bovine peripheral nerve. The obtained bovine parkin cDNA sequence was identical to that of human except a single nucleotide. Immunoblot analysis demonstrated the expression of parkin protein in bovine peripheral nerve. Immunohistochemical analysis demonstrated the localization of parkin in the axoplasm of myelinated nerve fibers, the Schwann cell cytoplasm and the Schwann cell outer membrane. Furthermore, fractionation analysis indicated the presence of two fractions of parkin in bovine peripheral nerve, the cytosolic fraction and the cell membrane-bound fraction. All together, these results point to diverse roles of parkin in not only the central but also the peripheral nervous system.

GDNF expression in human skeletal muscle

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related to TGF-P and are survival factors for sympathetic, sensory, and central nervous system neurons. GDNF transmits its signal primarily through a receptor complex containing the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)linked receptor, GDNFRa. NTN utilizes a receptor complex system that consists of Ret and another GPI-linked receptor, NTNRa. We have identified a mouse cDNA, termed GFRa-3, that encodes a putative GPI-linked receptor using the mouse expressed sequence tag database. At the protein level, mouse GFRa-3 is 35% identical to mouse GDNFRa and 36% identical to mouse NTNRa. Northern blot analysis showed that GFRu-3 is expressed in fetal mouse heart, brain, lung, and kidney and adult heart. These results indicate that the tissue distribution of GFRa-3 mRNA is different from that of GDNFRa or NTNRa mRNA, and suggest that GFRa-3 may function in differentiation of embryonic cells expressing its mRNA. The screening to isolate the ligand(s) for GFRa-3 is currently progressing.