ABSTRACT
CLP36, a member of the alpha-actinin-associated LIM protein (ALP)/enigma protein family, plays a role in neurite outgrowth in the peripheral nervous system. However, the underlying molecular mechanisms are not known. In this study, we performed yeast two-hybrid screening of an E18 mouse whole-body cDNA library with CLP36 as the bait and isolated palladin as a CLP36-binding protein. Palladin is an actin-binding protein and it was shown to have a role in the extension of cortical neurons. A coimmunoprecipitation study showed that CLP36 and palladin formed a complex in the dorsal root ganglion (DRG). In addition, CLP36 and palladin were colocalized in the neurites and cell bodies of primary DRG neurons. Furthermore, sciatic nerve transection increased the expression of both CLP36 and palladin mRNAs in DRG neurons, with the increase in CLP36 mRNA being more prominent. This implies that CLP36 has a more specific role in nerve regeneration than palladin. Our results suggest that CLP36 may interact with palladin to influence neurite outgrowth during sciatic nerve regeneration.
Subject(s)
Cytoskeletal Proteins/metabolism , Ganglia, Spinal/metabolism , Homeodomain Proteins/metabolism , Neurons/metabolism , Phosphoproteins/metabolism , Animals , Cells, Cultured , Cytoskeletal Proteins/genetics , Ganglia, Spinal/cytology , Homeodomain Proteins/genetics , LIM Domain Proteins , Male , Mice , Neurites/metabolism , Phosphoproteins/genetics , Protein Binding , RNA, Messenger/metabolism , Rats , Rats, Wistar , Regeneration , Sciatic Nerve/injuries , Sciatic Nerve/physiology , Transcription Factors , Two-Hybrid System TechniquesABSTRACT
CLP36, one of the alpha-Actinin Associated LIM Protein (ALP)/Enigma family proteins, has a wide tissue distribution, but little is known about its expression and role in the nervous system. We show here that CLP36 is expressed in sensory ganglia but not in the CNS of adult rats. In primary dorsal root ganglion (DRG) neurons, CLP36 is distributed in the soma and neurites with enrichment in the growth cones. CLP36 forms a complex with alpha-actinin and is localized to actin cytoskeleton. To examine the role of CLP36 in neuronal cells, we transfected PC12 cells with a series of CLP36 deletion mutants and found that over-expression of CLP36 PDZ domain affects neurite outgrowth. Reduction of CLP36 function in PC12 cells by RNA interference (RNAi) induced lamellipodial protrusions around cell periphery and activated growth-cone movements, resulting in an increase in the length and number of neurites. Similarly, inhibition of CLP36 in primary DRG neurons increased the rate of neurite-bearing cells. We also found that CLP36 is up-regulated in DRG neurons and facial motoneurons after nerve injury. These findings suggest that CLP36 serves as a scaffold to form a multiprotein complex that regulates actin cytoskeleton dynamics and plays a role in controlling neurite outgrowth.
Subject(s)
Gene Expression Regulation/physiology , Microfilament Proteins/metabolism , Nervous System/metabolism , Neurons/metabolism , Actin Cytoskeleton/metabolism , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cells, Cultured , Facial Nerve Injuries/metabolism , Facial Nerve Injuries/pathology , Ganglia, Spinal/cytology , Ganglia, Spinal/pathology , Ganglion Cysts , Gene Expression/physiology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Growth Cones/drug effects , Growth Cones/physiology , Immunoprecipitation/methods , LIM Domain Proteins , Microfilament Proteins/genetics , Mutation/genetics , Nerve Growth Factor/pharmacology , Nervous System/growth & development , Neurites/drug effects , Neurites/physiology , Neurons/classification , Neurons/cytology , Neurons/drug effects , PC12 Cells/cytology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Sciatic Neuropathy/metabolism , Sciatic Neuropathy/pathology , Transcription Factors , Transfection/methodsABSTRACT
Nogo receptors (NgR1, -2, and -3) and their ligands, i.e., myelin-derived neurite outgrowth inhibitor (Nogo)-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), have been considered to play pivotal roles in controlling axonal regeneration and neuronal plasticity. We show here that NgR1-3 mRNAs were differentially expressed exclusively in neurons situated in the telencephalon, diencephalons, and cerebellum, whereas we could not detect any NgR1-3 mRNA expression in the mesencephalon, pons, medulla oblongata, and spinal cord. On the other hand, Nogo-A mRNA was abundantly expressed in both neurons and oligodendrocytes throughout the central nervous system (CNS). MAG and OMgp mRNAs were also abundantly expressed in oligodendrocytes throughout the CNS. Interestingly, we did not detect NgR1-3 mRNAs in monoaminergic neurons in the substantia nigra, ventral tegmental area, locus caeruleus, and raphe nuclei, which are known to have high regenerative capacity. In addition, although neurons in the reticular thalamus and cerebellar nuclei are also known to show high capacity for regeneration, NgR1-3 mRNAs were not detected there. These data indicate that NgR1-3, Nogo-A, MAG, and OMgp mRNAs are differentially expressed in the rat CNS and suggest that the level of NgR1-3 expression in a neuron might determine its regenerative capacity.
Subject(s)
Central Nervous System/physiology , Myelin Proteins/genetics , Myelin-Associated Glycoprotein/genetics , Rats, Wistar/physiology , Receptors, Peptide/genetics , Age Factors , Animals , Central Nervous System/cytology , GPI-Linked Proteins , Gene Expression Regulation , Ligands , Male , Myelin Proteins/metabolism , Myelin-Associated Glycoprotein/metabolism , Myelin-Oligodendrocyte Glycoprotein , Neurons/physiology , Nogo Proteins , Nogo Receptor 1 , Oligodendroglia/physiology , RNA, Messenger/metabolism , Rats , Receptors, Cell Surface , Receptors, Peptide/metabolismABSTRACT
The purpose of this study was to investigate quantitatively the longitudinal temporal, spatial changes of the tumor necrosis factor-alpha (TNF) and interleukin-10 (IL-10) immunopositive cells during Wallerian degeneration and the following regeneration after crush injury in rat sciatic nerve using immunohistochemistry and enzyme linked immunosorbent assay (ELISA). The number of TNF-immunopositive cells reached its peak and increased significantly in all the segments distal to the crush site 3 days after injury. On Day 7, TNF-immunopositive cells decreased in all the segments distal to the crush site, and a significant decrease was observed 14 days after injury. From Day 21 to Day 56, there were no significant differences in the numbers of TNF-immunopositive cells. The average size of TNF immunopositive cells became significantly larger with degeneration. The number of IL-10-immunopositive cells decreases significantly 1 day after crush injury. IL-10-immunopositive cells increased on Day 3, returning to control levels. Seven days after injury, a significant increase in the number of IL-10-immunopositive cells was observed. There was also no significant difference in the number of IL-10-immunopositive cells beyond Day 14 except for a part of distal segments. The number of IL-10-immunopositive cells showed no significant differences in all the segments on Day 56. The protein levels of IL-10 measured by ELISA were similar to the result of immunohistochemistry. These results suggest that the significant change in IL-10 occurred prior to the significant change in TNF and that IL-10 may be the key to the change in TNF.
