Peripherin Is Tyrosine-Phosphorylated at Its Carboxyl-Terminal Tyrosine

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim⁻ cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim⁻ cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim⁻ cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.     

Evaluation of RDS/Peripherin and ROM1 as candidate genes in generalised progressive retinal atrophy and exclusion of digenic inheritance

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in the RDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate gene RDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.     

Intermediate Filament Disassembly in Cultured Dorsal Root Ganglion Neurons Is Associated with Amino-Terminal Head Domain Phosphorylation of Specific Subunits

Abstract: We previously reported that activation of protein kinase A in cultured rat dorsal root ganglion neurons, treated concomitantly with low concentrations of okadaic acid that selectively inhibit protein phosphatase-2A, enhanced the Triton X-100 solubility of neurofilament triplet proteins. We now show that peripherin and α-internexin follow the same fragmentation profile as the neurofilament subunits, consistent with the notion that all five cytoplasmic intermediate filament proteins in these neurons form an integrated filamentous network whose assembly can be modulated by protein kinase A. Similar to the situation previously observed for the light neurofilament subunit, there was a strong correlation between phosphorylation of the amino-terminal head domain of peripherin and filament fragmentation. In contrast, insignificant levels of ³²P were incorporated into α-internexin under conditions promoting disassembly, indicating that phosphorylation of this protein is not involved directly in filament fragmentation. The situation for the mid-sized neurofilament subunit (NFM) was not as clear-cut. Phosphopeptide mapping of NFM revealed many head and tail domain phosphorylation sites. However, changes in NFM head domain phosphorylation under conditions promoting filament disassembly were not as pronounced as for peripherin.     

Ultrastructural Studies of Carboxyl-Terminal Truncation Mutants of the Neuronal Intermediate Filament Protein Peripherin

Abstract: We have prepared carboxyl-terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per-C424) self-assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per-C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin-positive cells are transfected with Per-C424 or Per-C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles and clumps formed by Per-C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per-C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per-C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal ribosomal entry site, we prepared a construct that expresses Per-C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per-C356 by fluorescence-activated cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.     

Identification of a Peripherin Dimer: Changes During Axonal Development and Regeneration of the Rat Sciatic Nerve

Abstract: Western blotting of rat dorsal root ganglion (DRG) and sciatic nerve under nonreducing conditions revealed that a peripherin-specific antibody recognized a protein species of 116/130 kDa, pi 5.6, in addition to peripherin (56 kDa, pl 5.6). We showed that this 116/130 kDa protein is a disulfide dimer of peripherin, because it gave rise to a single protein band comigrating with peripherin under reducing conditions and yielded the same proteolytic pattern as peripherin upon N-chlorosuccinimide digestion. In addition, the immunological characteristics of the resulting peptides were identical to those of peripherin. We investigated the changes in peripherin monomer and dimer protein levels during axonal development and regeneration. During postnatal development, quantitative analysis of western blots of DRG proteins showed a significant increase in peripherin monomer (+52%) and dimer (+33%) levels from the day of birth [postnatal day 0 (PO)] to P7. The monomer levels remained high until P14 and then decreased so that at P21 and later ages, the monomer levels were similar to those observed at birth. In contrast, the dimer levels decreased continuously after P7, and in the adult, its level represented only 30% of the level at birth. Changes in [³⁵S]methionine incorporation into adult DRG proteins were studied during regeneration of axotomized sciatic axons. Quantitative analysis of proteins showed a strong increase in labeling of both peripherin monomer (+56%) and dimer (+88%) 7 days after the crush. These levels, which remained high until 28 days after the axotomy, had returned to normal 70 days post axotomy. Our results show that peripherin monomer and dimer greatly increase during DRG fiber development and regeneration, suggesting that the two forms are involved in the growth of axons.     

Synemin isoforms during mouse development: Multiplicity of partners in vascular and neuronal systems

The intermediate filament (IF) synemin gene encodes three IF proteins (H 180, M 150, L 41 kDa isoforms) with overlapping distributions. In the present study we analysed the mRNA and protein expression of each isoform in developing mouse embryos. Synemin M mRNA was present as early as E5 with vimentin and nestin. Synemin H was found later at E9 in the nervous system and mesodermic derivatives concomitantly with angiogenesis, somitogenesis and the migration of neural crest cells. Synemin L appeared later in neurons at E15. Furthermore, the synemin isoforms required different IF partners depending on the cell type to form filamentous structures. In endothelial cells, synemin H/M were found associated with vimentin and were absent in vimentin-null mice. In neurons of the peripheral nervous system of E15 embryos, synemin H/M or L were co-expressed with neurofilament, peripherin and internexin. In adult mice, our data support the existence of different subpopulations of neurons within the dorsal root ganglia: one composed of small neurons containing synemin H/M and peripherin, and another composed of large neurons containing synemin L and neurofilaments. Axons devoid of neurofilaments from mutant mice (NFHLacZ) showed an absence of the L isoform but contained H/M isoforms with peripherin.     

Transgenic mice expressing the <Emphasis Type="Italic">Peripherin-EGFP</Emphasis> genomic reporter display intrinsic peripheral nervous system fluorescence

The development of homologous recombination methods for the precise modification of bacterial artificial chromosomes has allowed the introduction of disease causing mutations or fluorescent reporter genes into human loci for functional studies. We have introduced the EGFP gene into the human PRPH-1 locus to create the Peripherin-EGFP (hPRPH1-G) genomic reporter construct. The hPRPH1-G reporter was used to create transgenic mice with an intrinsically fluorescent peripheral nervous system (PNS). During development, hPRPH1-G expression was concomitant with the acquisition of neuronal cell fate and growing axons could be observed in whole embryo mounts. In the adult, sensory neurons were labeled in both the PNS and central nervous system, while motor neurons in the spinal cord had more limited expression. The fusion protein labeled long neuronal processes, highlighting the peripheral circuitry of hPRPH1-G transgenic mice to provide a useful resource for a range of neurobiological applications.     

Transgenic Analysis of Rds/Peripherin N-Glycosylation

Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein rom1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N -glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with rom1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· rom1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N -glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N -glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Intermediate filament steady-state mRNA levels in amyotrophic lateral sclerosis

We have examined the steady-state levels of intermediate filament mRNA in amyotrophic lateral sclerosis using the RNAse protection assay (NFL, NFM, NFH; corrected against GAPDH) or by PCR (peripherin, alpha-internexin, nestin, and vimentin; corrected against beta-actin). Significant elevations of NFL and peripherin mRNA levels were observed within the ALS cervical and lumbar spinal cord, with all other IF mRNA levels being comparable between control and ALS cases. These findings suggest that disturbances in both NFL and peripherin expression, independently known to contribute to the generation of motor neuron dysfunction in transgenic mice, are evident in ALS.