Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies.

Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.     

Cloning of the cDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies.

The molecules essential to the continual morphogenesis and shedding of the opsin-containing disks of vertebrate photoreceptors are largely unknown. We describe a 37 kd protein, rom-1, which is 35% identical and structurally similar to peripherin/retinal degeneration slow (rds). Like peripherin, rom-1 is a retina-specific integral membrane protein localized to the photoreceptor disk rim. The two proteins are similarly oriented in the membrane, and each has a highly conserved (15/16 residues) cysteine- and proline-rich domain in the disk lumen. Although both rom-1 and peripherin form disulfide-linked dimers, they do not form heterodimers with each other, but appear to associate noncovalently. These results suggest both that rom-1 and peripherin are functionally related members of a new photoreceptor-specific protein family and that rom-1, like peripherin, is likely to be important to outer segment morphogenesis. The association of mutations in RDS with retinitis pigmentosa indicates that ROM1 is a strong candidate gene for human retinopathies.     

Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration

Peripherin/Rds is a tetraspanning membrane protein that has been implicated in photoreceptor outer segment morphogenesis and inherited retinal degenerative diseases. Together with the structurally related protein, Rom-1, it forms a complex along the rims of rod and cone disc membranes. We have compared the oligomeric structure of these proteins from nonreduced and dithiothreitol reduced membranes by velocity sedimentation, SDS-gel electrophoresis, immunoaffinity chromatography, and chemical cross-linking. Under reducing conditions peripherin/Rds and Rom-1 existed as homomeric and heteromeric core complexes devoid of intermolecular disulfide bonds. Under nonreducing conditions core complexes associated through intermolecular disulfide bonds to form oligomers. One intermediate-size oligomer contained monomers and disulfide-linked dimers of peripherin/Rds and Rom-1, while larger oligomers consisted only of disulfide-linked peripherin/Rds dimers when analyzed on nonreducing SDS gels. Consistent with this result, disc membranes contained twice as much peripherin/Rds as Rom-1. Peripherin/Rds individually expressed in COS-1 cells also formed disulfide-linked oligomers bridged through Cys-150 residues, whereas Rom-1 showed little tendency to form oligomers. These results indicate that peripherin/Rds and Rom-1 associate noncovalently to form multisubunit core complexes. Peripherin/Rds containing core complexes interact through specific intermolecular disulfide bonds to form oligomers which may play a crucial role in photoreceptor disc morphogenesis and retinal degenerative diseases.     

Peripherin/rds influences membrane vesicle morphology. Implications for retinopathies

Peripherin/rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. Natural mutations of the encoding gene result in degenerative retinal disorders, such as retinitis pigmentosa. The retinal degeneration slow (rds) phenotype, observed in mice, is considered to be an appropriate model for peripherin/rds-mediated retinitis pigmentosa. Associated abnormalities in the outer segment of photoreceptor cells have implicated peripherin/rds in some aspect of disc morphology, yet it remains unclear whether such morphological effects are the cause or the result of this condition. Here we present the first direct evidence to support a role for peripherin/rds in maintaining the flattened vesicle morphology characteristic of photoreceptor outer segments. In vitro expression yields a 36-kDa immunoreactive species, which is inserted into membranes and undergoes N-glycosylation, inter- and intramolecular disulfide bonding, and dimerization. Electron microscopy reveals that peripherin/rds flattens microsomal vesicles. This effect appears to be dependent on disulfide bond formation but not N-glycosylation. The inability of two pathogenic peripherin/rds mutants (P216L and C165Y) to flatten membrane vesicles implicates such mutations as the primary cause of the retinal degeneration observed in retinitis pigmentosa.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

The C terminus of peripherin/rds participates in rod outer segment targeting and alignment of disk incisures

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations.     

Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane

Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.     

Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone-dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous studies have revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation, we hypothesized that it is important for RDS function in cones and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein 1 (ROM-1) were found in N229S retinas. However, cone electroretinogram responses were decreased by 40% at 6 months of age. Because cones make up only 3–5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl^−/− background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl^−/− retinas, RDS and ROM-1 levels were decreased by ∼60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular versus intradiscal localization of the RDS glycan in cones versus rods.     

A two‐hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25‐interacting protein,SIP30

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up‐regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two‐hybrid screen on a mouse brain cDNA library using an assembly incompetent peripherin isoform, Per‐61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism. We focused on the interaction of Per‐61 and the constitutive isoform, Per‐58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor‐dependent exocytosis. We found that peripherin and SIP30 interacted through coiled‐coil domains and colocalized in cytoplasmic aggregates in SW13vim(?) cells. Interestingly, Per‐61 and Per‐58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking.

     

Uncoupling of photoreceptor peripherin/rds fusogenic activity from biosynthesis, subunit assembly, and targeting: a potential mechanism for pathogenic effects

Inherited defects in the RDS gene cause a multiplicity of progressive retinal diseases in humans. The gene product, peripherin/rds (P/rds), is a member of the tetraspanin protein family required for normal vertebrate photoreceptor outer segment (OS) architecture. Although its molecular function remains uncertain, P/rds has been suggested to catalyze membrane fusion events required for the OS renewal process. This study investigates the importance of two charged residues within a predicted C-terminal helical region for protein biosynthesis, localization, and interaction with model membranes. Targeted mutagenesis was utilized to neutralize charges at Glu(321) and Lys(324) individually and in combination to generate three mutant variants. Studies were conducted on variants expressed as 1) full-length P/rds in COS-1 cells, 2) glutathione S-transferase fusion proteins in Escherichia coli, and 3) membrane-associated green fluorescent protein fusion proteins in transgenic Xenopus laevis. None of the mutations affected biosynthesis of full-length P/rds in COS-1 cells as assessed by Western blotting, sedimentation velocity, and immunofluorescence microscopy. Although all mutations reside within a recently identified localization signal, none altered the ability of this region to direct OS targeting in transgenic X. laevis retinas. In contrast, individual or simultaneous neutralization of the charged amino acids Glu(321) and Lys(324) abolished the ability of the C-terminal domain to promote model membrane fusion as assayed by lipid mixing. These results demonstrate that, although overlapping, C-terminal determinants responsible for OS targeting and fusogenicity are separable and that fusogenic activity has been uncoupled from other protein properties. The observation that subunit assembly and OS targeting can both proceed normally in the absence of fusogenic activity suggests that properly assembled and targeted yet functionally altered proteins could potentially generate pathogenic effects within the vertebrate photoreceptor.     

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.     

Structural and Functional Analysis of the Native Peripherin-ROM1 Complex Isolated from Photoreceptor Cells

Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function.     

Function of <Emphasis Type="Italic">Drosophila mob2</Emphasis> in photoreceptor morphogenesis

The Drosophila photoreceptor is a highly polarized cell; a mature photoreceptor cell in Drosophila contains a photosensitive structure (the rhabdomere) and a supporting membrane (stalk) at its apical membrane. In a screen to isolate genes involved in determining stalk and rhabdomere formation, this study has identified the Drosophila mob2 (Dmob2) gene. Dmob2 belongs to a Mob1/phocein domain protein family whose functions are involved in polarized cell growth and asymmetric cell fate determination in yeast. To study the role of Dmob2 in photoreceptor development, we have raised an antibody against the Dmob2 protein. An immunocytochemical study has shown that Dmob2 is mainly localized in the apical membrane of photoreceptor cells during early development. As development proceeds, Dmob2 is gradually confined to the rhabdomere base of the photoreceptor cells. RNA interference (RNAi) for knockdown Dmob2 expression during eye development impairs rhabdomere formation. Our study further shows that the subcellular localization of phosphorylated Moesin and Crumbs in the developing photoreceptor cell is disrupted in Dmob2 RNAi flies. This work thus reports a novel function of Dmob2 in photoreceptor cell development.     

Mapping and retinal phenotype of the hugger mutation in the mouse

Hugger, hug, is a recessively expressed mutation in mice that features mildly abnormal locomotion, not yet explained, and a unique combination of developmental and degenerative retinal abnormalities. Analysis with the efficient MEV linkage testing stock established that hug is on mouse Chr 19 about 14 cM from th centromere, between the microsatellite markers D19Mit28 and D19Mit14. An abnormal retinal phenotype was recognized on the day of birth, when some retinal ganglion cells already lie in abnormal positions in the inner plexiform layer. By postnatal day 18 the number of neurons is reduced in all three cellular layers of the retina. Rod photoreceptor cells develop only rudimentory outer segments, and by 9 months of age, about 75% of the photoreceptor cells have completely disappeared. Similar photoreceptor cell abnormalities are seen in prph2 (formerly rds) homozygotes, which lack the peripherin/rds protein of the rod outer segments, but a mating of the respective homozygotes yielded normal progeny. Rom1, which codes for an outer segment protein similar to peripherin/rds, maps to a more proximal position on Chr 19. Received: 4 October 1996 / Accepted: 31 January 1997     

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.     

PXA Domain-Containing Protein Pxa1 Is Required for Normal Vacuole Function and Morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

A role for intermediate filaments in determining and maintaining the shape of nerve cells

To date, the functions of most neural intermediate filament (IF) proteins have remained elusive. Peripherin is a type III intermediate filament (IF) protein that is expressed in developing and in differentiated neurons of the peripheral and enteric nervous systems. It is also the major IF protein expressed in PC12 cells, a widely used model for studies of peripheral neurons. Dramatic increases in peripherin expression have been shown to coincide with the initiation and outgrowth of axons during development and regeneration, suggesting that peripherin plays an important role in axon formation. Recently, small interfering RNAs (siRNA) have provided efficient ways to deplete specific proteins within mammalian cells. In this study, it has been found that peripherin-siRNA depletes peripherin and inhibits the initiation, extension, and maintenance of neurites in PC12 cells. Furthermore, the results of these experiments demonstrate that peripherin IF are critical determinants of the overall shape and architecture of neurons.     

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.     

Late onset of motor neurons in mice overexpressing wild-type peripherin

Peripherin, a type III intermediate filament (IF) protein, upregulated by injury and inflammatory cytokines, is a component of IF inclusion bodies associated with degenerating motor neurons in sporadic amyotrophic lateral sclerosis (ALS). We report here that sustained overexpression of wild-type peripherin in mice provokes massive and selective degeneration of motor axons during aging. Remarkably, the onset of peripherin-mediated disease was precipitated by a deficiency of neurofilament light (NF-L) protein, a phenomenon associated with sporadic ALS. In NF-L null mice, the overexpression of peripherin led to early- onset formation of IF inclusions and to the selective death of spinal motor neurons at 6 mo of age. We also report the formation of similar peripherin inclusions in presymptomatic transgenic mice expressing a mutant form of superoxide dismutase linked to ALS. Taken together, these results suggest that IF inclusions containing peripherin may play a contributory role in motor neuron disease.