NIH Public Access 1,2 1 Asok Kumar1,2 Corrinne M ...cdr.rfmh.org/Publications/PDFs_Nixon/Yuan 2012 J...

Peripherin Is a Subunit of Peripheral Nerve Neurofilaments:Implications for Differential Vulnerability of CNS and PNS Axons

Aidong Yuan1,2, Takahiro Sasaki1, Asok Kumar1,2, Corrinne M. Peterhoff1, Mala V. Rao1,2,Ronald K. Liem4, Jean-Pierre Julien5, and Ralph A. Nixon1,2,3

1Center for Dementia Research, Nathan Kline Institute, Orangeburg, New York 109622Department of Psychiatry, New York University School of Medicine, New York, New York 100163Department of Cell Biology, New York University School of Medicine, New York, New York100164Department of Pathology, College of Physicians and Surgeons, Columbia University, New York,New York 100325Centre de Recherche du Centre Hospitalier de l’Université Laval, Département d’anatomie etphysiologie de l’Université Laval, 2795 boul. Laurier, Québec G1V 4G2, Canada

AbstractPeripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease,coexists with the neurofilament triplet proteins (NFL, NFM, and NFH) but has an unknownfunction. The earlier peak expression of peripherin than the triplet during brain development andits ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, havesupported a widely held view that peripherin and neurofilament triplet form separate filamentsystems. Here, we demonstrate, however, that despite a postnatal decline in expression, peripherinis as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry withthese subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins insciatic axons and co-localizes with NFL on single neurofilament by immunogold electronmicroscopy. Peripherin also co-assembles into a single network of filaments containing NFL,NFM, NFH with and without α-internexin in quadruple- or quintuple-transfected SW13 vim (−)cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons.Moreover, peripherin mutations has been shown to disrupt the neurofilament network intransfected SW13 vim(−) cells. These data show that peripherin and the neurofilament proteins arefunctionally interdependent. The results strongly support the view that rather than forming anindependent structure, peripherin is a subunit of neurofilaments in the adult PNS. Our findingsprovide a basis for its close relationship with neurofilaments in PNS diseases associated withneurofilament accumulation.

Address correspondence to: Aidong Yuan, M.D., Ph.D. or Ralph A. Nixon, M.D., Ph.D., Center for Dementia Research, Nathan KlineInstitute, New York University School of Medicine, 140 Old Orangeburg Rd., Orangeburg, NY 10962, Tel.: (845) 398-5450; Fax:(845) 398-5422, [email protected] or [email protected]. Sasaki’s present address: Department of Peripheral Nervous System, National Institute of Neuroscience, National Center ofNeurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187-8502, Japan.

Author contributions: A.Y. designed research; A.Y., T.S., A.K., and C.M.P performed research; A.Y., M.V.R, R.K.L, J-P.J. andR.A.N. analyzed data; A.Y. and R.A.N. wrote the paper.

No conflict of interest

NIH Public AccessAuthor ManuscriptJ Neurosci. Author manuscript; available in PMC 2012 December 20.

Published in final edited form as:J Neurosci. 2012 June 20; 32(25): 8501–8508. doi:10.1523/JNEUROSCI.1081-12.2012.

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Keywordsperipherin; neurofilament; intermediate filament; cytoskeleton; peripheral nerve; ALS; PNS

IntroductionNeurofilaments in the mature CNS are composed of four subunits, NFH, NFM, NFL and α-internexin (Yuan et al., 2006). In most adult peripheral nerves, however, α-internexin ismarkedly down-regulated during embryonic development to hardly detectable levels and itsdisappearance from the PNS coincides with the appearance of another intermediate filament(IF) protein, peripherin (Escurat et al., 1990). Following its discovery as a 57kDa Triton-insoluble protein (Portier et al., 1982; Tatemoto et al., 1982; Portier et al., 1983b; Portier etal., 1983a), peripherin was established as a type III IF protein based on its structuralproperties and ability to assemble into filaments by itself or together with eitherneurofilament proteins or vimentin (Portier et al., 1983a; Parysek and Goldman, 1987;Leonard et al., 1988; Thompson and Ziff, 1989; Cui et al., 1995; Ho et al., 1995; Athlan andMushynski, 1997). Although its neuronal specificity and abundance in axons led earlyinvestigators to suspect that peripherin, like α-internexin, may be a subunit ofneurofilaments (Portier et al., 1983a), little evidence has been brought to bear on thispossibility (Barry et al., 2007; Mclean and Robertson, 2011). Moreover, other studies havesupported the view that peripherin forms a separate filament system (Ferri et al., 1990; Troyet al., 1990; Wong and Oblinger, 1990; Beaulieu et al., 1999) although the evidence isindirect. For example, peripherin can form homopolymers (Cui et al., 1995; Ho et al., 1995),unlike neurofilament triplets which are obligate heteropolymers (Ching and Liem, 1993; Leeet al., 1993). Also, peripherin gene expression is highest in late embryonic and earlypostnatal brain and declines somewhat before expression of triplet protein sharply rise (Kostet al., 1992). Peripherin and neurofilament gene expression is also regulated differentially innerve regeneration (Troy et al., 1990; Wong and Oblinger, 1990; Belecky-Adams et al.,1993). These studies have lead to a prevailing view that the neurofilaments in the maturePNS are composed of three subunits (Elder et al., 1998; Yuan et al., 2006) although thephysiological roles of peripherin, in fact, remain incompletely understood.

Beyond its important neurobiological implications, understanding peripherin’s function andrelationship to neurofilaments takes on additional significance in light of the recentidentification of peripherin mutations in amyotrophic lateral sclerosis (ALS) (Gros-Louis etal., 2004; Leung et al., 2004; Corrado et al., 2011), a neurodegenerative disorder in which apathological hallmark is the abnormal accumulation of neurofilaments in motor neurons(Hirano et al., 1984; Kondo et al., 1986; Munoz et al., 1988; Mizusawa et al., 1989; Stronget al., 2001).

To investigate the possibility that peripherin serves a role in the mature PNS analogous to α-internexin in CNS, we studied the behavior and distribution of peripherin underphysiological conditions in vivo and in other conditions where neurofilament behaviors werealtered by genetically deleting specific neurofilament subunits. Our data identify importantnew structural and functional relationships between peripherin and neurofilament triplets.Moreover, by applying criteria previously used to establish the triplets as neurofilamentsubunits and α-internexin in CNS (Yuan et al., 2006), we obtained strong evidence thatperipherin is a fourth subunit of neurofilaments in the mature PNS. Our studies reconcileprevious observations on peripherin, including the similar behavior of peripherin and theneurofilament triplet proteins in pathological states, and they reveal a potential basis for thedifferential vulnerability of CNS and PNS neurons in neurological diseases.

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J Neurosci. Author manuscript; available in PMC 2012 December 20.

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Materials and MethodsGeneration of knockout animals

Adult male or female mice of the C57BL/6J strain, aged 3 – 12 months at the time ofsacrifice, were used in all experiments. Mice were housed at 23°C on a 12-h light-dark cycleand were maintained on Lab Chow (Purina Mills, Gray Summit, MO) supplied at libitum.Productions of NFL knockout (LKO) and peripherin knockout have been previouslydescribed (Zhu et al., 1997; Lariviere et al., 2002). All experimental protocols wereapproved by the New York University and Nathan Kline Institute IACUC Committees underthe guidelines of the Institutional Animal Care and Use Committee of the United States.

SDS-PAGE and immunoblot analysisProtein concentrations were determined with bicinchoninic acid (BCA) assay (Sigma-Aldrich, St. Louis, MO). SDS-PAGE was performed according to Laemmli (Laemmli,1970). Separated proteins were transferred to nitrocellulose membranes (Millipore, MA).Blots were probed with primary antibodies, followed by an alkaline phosphatase-conjugatedsecondary antibody (12500-fold dilution, DAKO, Glostrup, Denmark). The reaction wasdeveloped using a BCIP/NBT phosphatase system (KPL, Gaithersburg, MD) as describedpreviously (Yuan et al., 1997). Sciatic nerves from LKO and wild-type controls werehomogenized in 500μl μl of buffer (SDS buffer) containing 25 mM sodium phosphate (pH7.2), 5 mM EGTA, 1% SDS, and 1 mM phenylmethylsulfonyl fluoride and proteinconcentration was measured with the BCA assay (Sigma-Aldrich, St. Louis, MO). Loadingonto gels was normalized to total protein of the nerves. Alternatively, a specific length ofsciatic nerve segment (6-mm) from each mouse was homogenized in 125μl of buffercontaining 25 mM sodium phosphate (pH 7.2), 5 mM EGTA, 1% SDS, and 1 mMphenylmethylsulfonyl fluoride. Three μl of the nerve extract were loaded into individuallanes of SDS-PAGE gels. Blots were probed with peripherin antibodies against totalperipherin. For the experiment to estimate the relative amount of neurofilament proteins inthe mature nervous system, optic pathways (about 1.5 mg wet weight) or similar amount ofcorpus callosum were homogenized in 250μl of SDS buffer while sciatic nerves (about 4.5mg wet weight) or similar amounts of spinal cord were homogenized in 500μl μl of SDSbuffer and processed as above for immunoblotting with specific antibodies.

Estimation of peripherin content in sciatic nerveTotal protein extracts from 24 mm sciatic nerve were homogenized in 250μl of buffercontaining 25 mM sodium phosphate (pH 7.2), 5 mM EGTA, 1% SDS, and 1 mMphenylmethylsulfonyl fluoride, and protein concentration was measured with BCA assay(Sigma-Aldrich, St. Louis, MO). One-dimensional 7.5, 10 or 12% SDS-PAGE gels wereused and the PAGE-separated proteins were transferred to nitrocellulose membranes andimmunoblotted using an alkaline-phosphatase-conjugated indirect 2° antibody procedure.Major proteins were identified using appropriate antibodies. Purified recombinant peripherin(tagged with 26kDa GST)(Novus Biologicals, Littleton, CO) or purifed NFL protein frommouse spinal cords (Rao et al., 2011) were used as a standard for protein estimation byquantitative immunoblotting. Sciatic nerves were also homogenized in 250 μl ofcytoskeleton extraction buffer (50 mM Tris-HCl, pH 6.8, 1% Triton X-1000, 10% glycerol,0.15 M NaCl, 2 mM EDTA, 2 mM PMSF, 50 μg/ml leupeptin, 2.5 μg/ml aprotinin). Thehomogenate was centrifuged at 15,000 g for 30 min at 4°C to isolate Triton-soluble andTriton-insoluble particulate fractions. Gels were also stained with Coomassie Brilliant BlueR in 50% methanol/10% acetic acid.

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Antibodies usedAntibodies used were mAbs to NFL (NR4), NFM (NN18), NFH (N52) (Sigma ChemicalCo.) and α-internexin (MAB5224, Chemicon Int.), respectively. Antibodies also used werepolyclonal antibodies to α-internexin (AB5354, Millipore, MA), NFL (AB9568, Millipore,MA), NFM (prepared using purified mouse NFM proteins as antigen) and NFH C-terminal12 aa (Xu et al., 1993). Antibodies also used were mAbs to peripherin (MAB1527,MAB5380-7C5, Chemicon Int) (MAB8G2, Sigma Chemical Co) and polyclonal antibody toperipherin (Chemicon Int).

Estimation of stoichiometry of peripherin and NFL along sciatic nerveConsecutive 3-mm segments were cut from 24 mm sciatic nerves away from spinal cord andhomogenized in 125 μl of cytoskeleton extraction buffer (50 mM Tris-HCl, pH 6.8, 1%Triton X-1000, 10% glycerol, 0.15 M NaCl, 2 mM EDTA, 2 mM PMSF, 50 μg/mlleupeptin, 2.5 μg/ml aprotinin). The homogenate was centrifuged at 15,000 g for 30 min at4°C to isolate Triton-soluble and Triton-insoluble particulate fractions. The pellets were thendissolved in sample preparation buffer (1% SDS, 10% glycerol, 10% beta-mercaptoethanol,250 mM Tris, pH 6.8) and heated for 10 min. Equal volumes were loaded onto each lane andthe PAGE-separated proteins were transferred to nitrocellulose membranes andimmunoblotted with anti-peripherin or anti-NFL antibodies. The molar stoichiometriesbetween peripherin and NFL along the sciatic nerve were estimated by computerdensitometry using NIH image as described before (Yuan et al., 2006).

Immunoelectron microscopyPeripherin null and wild-type mice, anesthetized with isoflurane, were perfusedintracardially with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 followed byfixation of the removed sciatic nerves for 4 hrs in fresh fixative on ice. Ultrathincryosections were prepared as described for postembedding immunostaining (Zheng et al.,2004). Peripherin was detected using rabbit polyclonal antibody (Chemicon International),followed by a 12-nm gold conjugated goat anti-rabbit antibody. NFL was detected using amouse monoclonal antibody (NR4, Sigma), followed by 6-nm gold conjugated goat anti-mouse. Sections were observed and photographed on a JEOL 1200 EXII transmissionelectron microscope. For alternative pre embedding immuno-EM, 50μ vibratome sectionsare incubated with polyclonal anti-peripherin or monoclonal anti-NFL and processed fordouble immunogold-silver labeling as described (Verkade et al., 1997; Yi et al., 2001).Differentiation of the double labeling was achieved by incubating with one ultrasmall goldconjugate (0.6-nm gold conjugated goat anti-rabbit antibody), followed by silverenhancement, and then incubating with the second ultrasmall gold conjugate (0.6-nm goldconjugated goat anti-mouse antibody), followed by additional silver enhancement. Thisresulted in two groups of silver-enhanced particles: smaller particles enhanced once andlarger particles enhanced twice. After fixing, post fixing in Osmium, and routine processingfor electron microscopy, ultrathin sections are stained with alcoholic uranyl acetate beforeimaging. The grids were photographed on a JEOL 100 cx electron microscope operated at80 kV.

Immunofluorescence of SW13vim(−) cellsSW13vim- cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10%fetal bovine serum (FBS) and 100 U/ml penicillin and 0.1 mg/ml streptomycin. Transfectioninto SW13- cells was performed using Lipofectamine 2000 (Invitrogen, Carsbad, CA)according to the manufacturer’s instructions. Immunofluorescence staining of SW13vim(−)cells were performed as described previously (Sasaki et al., 2006; Yuan et al., 2009). Theexpression plasmid of mouse NFL, NFM and NFH were constructed by cloning into

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pcDNA3.1. The expression plasmid of rat α-internexin (pRSVi-α)and rat peripherin(pRSVi-per) were used as previously described (Ching and Liem, 1993; Ho et al., 1995).NFL was probed with rabbit polyclonal antibody NA1214 (500-fold dilution, Affiniti,Nottingham, UK). NFM was probed with mouse monoclonal antibody NN18 (500-dilution,Sigma-Aldrich, St. Louis, MO) or antiNF-M rabbit polyclonal antibody (200-dilution)prepared using purified mouse NFM proteins as antigen. NFH was probed with mousemonoclonal antibody N52 (2000-fold dilution) or anti-NFH rabbit polyclonal antibodyraised against the COOH-terminal 12 amino acids of mouse NFH (2000-fold dilution) (Xu etal., 1993), and rat α-internexin was probed with anti-α-internexin mouse monoclonalantibody MAB5224 (200-dilution). Rat peripherin was probed with anti-peripherin mousemonoclonal antibody MAB5380 (100-dilution) (Clone 7C5, Millipore, Billerica, MA).Images were observed under an LSM 510 laser scanning confocal microscope (Carl ZeissInc., Oberkochen, Germany).

ResultsStable stoichiometry of peripherin and NFL in sciatic axonal neurofilaments

In mouse sciatic axons, peripherin was identified on SDS gels as a 57 kDa protein that isexclusively Triton-insoluble (Figure 1A, B) and recognized by anti-peripherin antibody onimmunoblots (Figure 1C). Since the 57kDa peripherin band is contaminated by anotherTriton-insoluble protein (here we name it p57) (Figure 1D, E), we choose not to useCoomassie-blue staining to estimate peripherin content in sciatic nerves. Quantitativeimmunoblotting with purified peripherin and NFL revealed that peripherin constituted 1.83± 0.21% (Mean ± SD, n = 8) of total protein in sciatic nerve (Figure 1F, H), whereas NFLconstituted about 6.88 ± 0.90 % (Mean ± SD, n = 8) (Figure 1G, H). Neurofilamentsdecrease in number proximally to distally along sciatic axons (Schlaepfer and Bruce, 1990).Additional immunoblots demonstrated that peripherin and NFL display the same degree ofproximal to distal decline in content, maintaining a constant ratio along the sciatic axons(Figure 2, Mean ± SD, n = 3).

Ultrastructural colocalization of peripherin and NFL on single neurofilament in sciaticaxons

To determine if peripherin and NFL co-exist on the same neurofilament in mouse sciaticaxons, we carried out ultrastructural colocalization studies of these two proteins usingpolyclonal anti-peripherin and monoclonal anti-NFL antibodies and double immunogoldlabeling. Thin sections of paraformaldehyde-fixed sciatic nerve were incubated with rabbitanti-peripherin polyclonal antibody and mouse anti-NFL monoclonal antibody (NR4) andthen stained with goat anti-rabbit and anti-mouse IgG conjugated with 12–nm and 6-nm goldparticles, respectively. When rabbit anti-peripherin and mouse anti-NFL antibodies wereincubated with the sections, the respective immunogold conjugated secondary antibodiesdetected both peripherin and NFL associated with the same 10nm filaments (Figure 3C, D)and the specificity of this interaction was confirmed by the negligible labeling of peripherinin peripherin knockout animals (Figure 3E). Co-localization of peripherin and NFL on singleneurofilament in sciatic nerve was also demonstrated by double immunogold-silver labelingprocedure (Figure 4A–D) and the specificity of this interaction was also confirmed by thenegligible labeling of peripherin in peripherin knockout animals (Figure 4E).

Quantitative immunoblot measurements (ratios) of peripherin, α-internexin, NFL, NFM andNFH in the corpus callosum, optic nerve, spinal cord and sciatic nerve of mature mice usingphosphorylation-independent antibodies highlighted the considerable regional heterogeneityof subunit stoicheiometry. Peripherin exhibited a stoichiometric relationship complementaryto that of α-internexin in neurofilaments with high levels of peripherin in sciatic nerve and

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low but not absent levels in CNS regions (Figure 5). By contrast, α-internexin wascomparably abundant in corpus callosum, optic nerve and spinal cord, but barely detectablein sciatic nerve. Consistent with growing evidence that NFM is the key subunit regulatingNF transport. NFM levels in optic nerve, spinal cord and sciatic nerve, were comparable inCNS and PNS regions. Similarly, NFL was comparably distributed across PNS and CNStissue but less abundant in corpus callosum, where NF numbers and axon calibers are thelowest among these regions. Conversely, NFH proportions were most variable acrossregions and correlated generally with the average calibers and abundance of NFs in the fourfiber tracts (Figure 5).

Peripherin co-assembles with other neurofilament subunit proteins into a single filamentnetwork

Previous studies have shown that all peripherin can co-assemble with NFL or NFL plusNFH in double-or triple-transfected SW13vim(−) cells to form the same neurofilamentnetwork (Beaulieu et al., 1999). It is not known, however, if peripherin self-assembles intoits own separate filamentous network, when all five proteins (peripherin, α-internexin, NFH,NFM and NFL) are present in the same cell as in the immature PNS (Chiu et al., 1989), orcan form complete neurofilaments. SW13vim(−) cells lacking cytoplasmic intermediatefilaments were, therefore, quintuple-transfected with constructs that expressed peripherin, α-internexin, NFH, NFM and NFL and were immunostained with mouse monoclonalantibodies to peripherin, α-internexin or NFL and rabbit polyclonal antibodies to α-internexin, NFL, NFM and NFH. Double immunofluorescence analyses with pairs of theseantibodies confirmed that all five proteins co-assembled into a single network of filaments(Figure 6). Under conditions where all four proteins (peripherin, NFH, NFM and NFL)coexist in the same cell, such as in the mature PNS, we also observed that all peripherin co-assembles with all three neurofilament proteins into a single filamentous network rather thanforming its own separate filamentous network. In immunofluorescence studies similar tothose above, SW13vim(−) cells expressing peripherin, NFH, NFM and NFL co-assembledinto a single network of filaments (Figure 7).

Dependence of peripherin on NFL in vivoWe next examined quantitatively how deleting NFL influences the levels of peripherin. Thesteady-state level of peripherin in sciatic nerves of 3-month-old mice was decreased to 28.08± 9.38% of wild type level in the absence of NFL (Mean ± SD, n = 4) whereas those ofNFM and NFH were decreased to 4.70 ± 1.95% (Mean ± SD, n = 4) and 14.00± 3.00% %(Mean ± SD, n = 4), respectively (Figure 8A, B). The level of vimentin was not significantlyaltered in NFL-deleted sciatic nerves (not shown). Electron microscopy studies confirmedthe scarcity of neurofilaments in sciatic axons from 3-month-old LKO mice (Figure 8C, C1,D, D1), indicating that peripherin can not self-assemble efficiently into filaments withoutNFL in vivo and neurofilament subunits accumulated in cell bodies (Williamson et al.,1998). Peripherin also accumulated slightly in some of the motor and sensory cell bodies inNFL deleted mice (Figure 9).

DiscussionIn this study, we demonstrate, by all criteria previously used to establish the neurofilament“triplet proteins” as subunits of neurofilaments, that peripherin is a fourth subunit of PNSneurofilaments. Although peripherin is a neuronal IF protein based on its co-assembly invitro with neurofilament proteins (Beaulieu et al., 1999), this property does not establish itas a neurofilament subunit. Nestin and vimentin are also neuronal IF proteins but are notneurofilament subunits. Moreover, neurofilament proteins co-assemble in vitro with other IFproteins, such as vimentin and keratin, which are clearly not neurofilament subunits

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(Monteiro and Cleveland, 1989; Carter et al., 1997). Our results, coupled with earlierfindings on α-internexin, establish that NFs in mature CNS and PNS neurons are foursubunit polymers.

Peripherin co-purified with neurofilament triplets from Triton-insoluble fractions in amountscomparable to those of the other subunits and exhibited a stable stoichiometry with NFLalong mature PNS axons where neurofilament number declines distally. We alsodemonstrated in quintuple-transfected SW13vim (−) cells, for the first time, that peripherinco-assembles into a single filamentous network with all four neurofilament proteins, as seenin immature PNS (Chiu et al., 1989) or with neurofilament triplets, as seen in the maturePNS. Moreover, peripherin co-localizes with NFL on the same neurofilament and in a molarratio of 3.5:1 (NFL: peripherin) in isolated neurofilaments from adult sciatic nerve. Based onmolar ratios of neurofilament subunits isolated from bovine spinal cord are 4:2:1(NFL:NFM:NFH) (Scott et al., 1985), we calculate that the molar ratio of the quadruplets inisolated neurofilaments from sciatic nerve is about 4:2:1:1 (NF-L:NF-M:peripherin:NF-H).A functionally meaningful association of peripherin with the neurofilament triplet in vivowas strongly supported by evidence that peripherin levels and filaments are dramaticallyreduced in adult sciatic nerves in the absence of NFL. Our findings are consistent withreports that peripherin co-assembles with NFL in SW13vim(−) cells (Beaulieu et al., 1999)and that peripherin mutations disrupt the neurofilament network in SW13 vim(−) cells(Parysek et al., 1991; Leung et al., 2004). Peripherin and neurofilament proteins aretransported at the same rate (Chadan et al., 1994) and are present in the same filament(Parysek et al., 1991; Yan et al., 2007).Moreover, overexpressing an NFH- LacZ fusionprotein in mice induces peripherin to aggregate with the neurofilament triplet in neuronalperikarya (Eyer and Peterson, 1994).

In the absence of NFL in sciatic nerve, only 4.7% of NFM were detected. This is in sharpcontrast to the optic nerve where 50% of the NFM still remains after NFL is deleted (Yuanet al., 2003). This difference is likely due to the presence of abundant α-internexin in opticaxons since further deletion of α-internexin in the absence of NFL and NFH abolishes NFMtransport and NFM is undetectable (not shown). These results highlight the much greaterability of the type IV IFs α-internexin and NFM to partner together than peripherin, a typeIII IF protein, as previous findings suggest (Ching and Liem, 1993; Beaulieu et al., 1999).Although deletions of both NFH and NFM significantly affect α-internexin, deletions ofboth NFH and NFL have little effect on α-internexin because 50% NFM is still present andcan partner with the α-internexin (Yuan et al., 2003). Similarly, the remaining peripherincould partner with remaining NFM and NFH to form oligomeric structures, as inSW13vim(−) cells (Beaulieu et al., 1999).

Peripherin pathology has been increasingly reported in neurodegenerative diseasesassociated with neurofilament accumulation or mislocation. Our evidence that peripherin isa neurofilament subunit in adult PNS provides a basis for this close association. Peripherinmutations in the rod domain (D141Y or R133P) cause ALS (Leung et al., 2004; Corrado etal., 2011), possibly through disruption of neurofilament networks (Parysek et al., 1991;Leung et al., 2004). In one ALS case, a pathogenic peripherin gene mutation was associatedwith distinctive large perikaryal aggregates containing peripherin and neurofilament triplet(Leung et al., 2004). A frameshift deletion in the peripherin gene has also been shown todisrupt neurofilament assembly in a subgroup of cases of ALS (Gros-Louis et al., 2004).Peripherin and neurofilament protein also coexist in spinal spheroids of ALS (Corbo andHays, 1992). Peripherin overexpression (4–7 fold) in transgenic mice slows neurofilamenttransport (Millecamps et al., 2006) and promotes late onset death of motor neurons(Beaulieu et al., 1999). The axonal transport of peripherin and NF triplet proteins is similarly

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impaired by beta-beta′-iminodipropionitrile (Griffin et al., 1978) or aluminum (Bizzi et al.,1984).

Interestingly, mutations of peripherin, NFL and NFH, subunits most abundant in the PNS,are associated primarily with peripheral nerve disorders such as Charcot-Marie-Toothdisease (CMT) and ALS (Braissant, 2007; Yum et al., 2009) whereas dysfunction of α-internexin is associated primarily with central nervous system disorders such asneurofilament inclusion disease, a form of frontal temporal dementia (Cairns et al., 2004).Mutations of NFM, a subunit abundant in both PNS and CNS axons, are associated withboth peripheral and central nervous system disorders, such as ALS and Parkinson’s disease(Braissant, 2007). Thus the associations of particular neurofilament subunits with peripheralor central nervous system disease match the relative abundance of the subunit in the matureCNS or PNS. Our results suggest that differences in neurofilament subunit compositionbetween the CNS and PNS should be considered as a possible contributing factor to thedifferential vulnerability of these neural systems in pathological states.

AcknowledgmentsThis work was supported by National Institutes on Aging Grant AG05604 (R.A.N). We thank Nicole Piorkowskifor manuscript preparation; Arthur Saltzman for histological assistance, Veeranna for purified NFL and TimoMeerloo (University of California, San Diego) for his help with post-embedding immuno-EM.

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Figure 1. Peripherin is as abundant as the NF triplet in the adult PNS and exists in a relativelyfixed stoichiometry with these subunitsPeripherin was identified as one of the major proteins in the sciatic nerve of 3-month-oldmice by Coomassie-blue staining (A) and is Triton-insoluble (B). The peripherin protein wasrecognized by anti-peripherin antibody on immunoblot (C). The 57kDa peripherin bandstained by Coomassie-blue is contaminated by another Triton-insoluble protein (p57) asevidenced by its presence in peripherin knockout mice (D) and peripherin deletion wasconfirmed by immunoblotting with specific anti-peripherin antibody (E). Measurement ofperipherin and NFL in sciatic nerve homogenates by quantitative immunoblotting (F, G).Purified peripherin with a 26kDa GST tag or purified NFL was used as standard in separateimmunoblots and incubated with either anti-peripherin or anti-NFL. In F, Lanes 1, 2, 3:purified peripherin, 40, 80 and 160 ng, respectively. In G, Lanes 1, 2, 3: purified NFL, 9, 18and 36 ng, respectively. Lanes 4–11, homogenates from 8 different sciatic nerves of 8 mice.Peripherin is as abundant as the NF triplet in the adult PNS and exists in a relatively fixedstoichiometry with these subunits. The molar ratio is estimated to be 3.5:1 (NFL:peripherin)based on quantitative immunoblotting (H). Neurofilament triplets were identified byimmunoblotting with specific antibodies and also by their disappearance in Triton-insolublefractions from corresponding knockout mice (NFH knockout, NFM knockout and NFLknockout). Note that the 68kDa Triton-soluble protein (Figure 1B) which contaminates NFLin homogenates (Figure 1A) was previous identified as serum albumin (Soifer et al., 1981).Per, peripherin, Pel, pellet; sol, soluble; wt, wild-type; pko, peripherin konockout.

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Figure 2. Distribution of peripherin and NFL along the sciatic nerveTriton-insoluble fractions from consecutive 3-mm segments of the sciatic nerve wereseparated by SDS-PAGE and transferred to nitrocellulose membranes and then probed byanti-peripherin and anti-NFL antibodies (A). Analyses of these immunoblots demonstratethat peripherin and NFL show the same proximal to distal distribution (B) and maintain aconstant ratio along the sciatic nerve (C). Per, peripherin.

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Figure 3. Ultrastructural colocalization of peripherin and NFL on the same neurofilament insciatic nerve by post-embedding immuno-EMParaformaldehyde-fixed samples were incubated with rabbit anti-peripherin or mouse anti-NFL antibodies and probed with goat anti-rabbit IgG and goat anti-mouse IgG conjugated to12nm and 6nm gold beads. As expected, for the immunodection of peripherin in normalmice (A), large numbers of 12nm gold particles are aligned with most 10-nm filaments inthe axon. (B) Higher magnification shows decoration of single filaments by 12nm goldparticles conjugated to anti-peripherin antibody. (C) Linear arrays of two sizes of goldparticles (12nm for peripherin and 6nm for NFL) decorate 10-nm filaments in the axon. (D)Higher magnification shows that gold particles of two sizes overlie a single filament in thebackground. Arrowheads point to 12-nm particles (peripherin) and arrows to 6-nm ones(NFL). (E) Negligible numbers of 12nm gold particles (peripherin) are detected inperipherin knockout mice while 6nm gold particles (NFL) are still present. Bars, 150nm inA; 40nm in B; 100nm in C; 30nm in D; 80nm in E.

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Figure 4. Ultrastructural colocalization of peripherin and NFL on the same neurofilament insciatic nerve by pre-embedding immuno-EMParaformaldehyde-fixed samples were incubated with rabbit anti-peripherin and mouse anti-NFL antibodies and probed with goat anti-rabbit IgG and goat anti-mouse IgG conjugated to0.6nm gold beads. Single (for anti-mouse IgG)- or double (for anti-rabbit IgG)-silverenhancement of gold particles resulted in irregular-shaped electron-dense particles thatcould be distinguished by their size. As expected, for the immunodetection of peripherin andNFL in normal mice (A), linear arrays of two sizes of gold particles (large for peripherin andsamll for NFL) decorate most 10-nm filaments in the axon and negligible numbers aredetected in peripherin knockout mice (E). Higher magnification shows that gold particles oftwo sizes overlie a single filament in the background (B, C, D). Arrows point to smallparticles (NFL) and arrowheads to large ones (peripherin). Scale bars, 100 nm in A; 60 nmin B; 40 nm in C; 50 nm in D; 200 nm in E.

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Figure 5. The distribution of peripherin is complementary to that of α-internexin in the maturenervous systemThe relative amount of peripherin, α-internexin, NFL, NFM and NFH in the mature nervoussystem (represented by optic pathway, corpus callosum, spinal cord and sciatic nerve) wasestimated by SDS-PAGE and immunoblotting (examples shown). Data are presented frommultiple experiments (Mean ± SEM, n = 4) as column graphs normalized to spinal cord.Note that the distribution of peripherin is complementary to that of α-internexin. opn, opticpathway; cc, corpus callosum; sc, spinal cord; sn, sciatic nerve; α-int, α-internexin; Per,peripherin.

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Figure 6. Co-assembly of peripherin with NF triplets and α-internexin into single filamentnetworkSW13vim(−) cells were quintuple-transfected with constructs that expressed peripherin,NFL, NFM, NFH and α-internexin and were immunostained with pairs of antibodies. Eachset of 3 panels across represents the single immunolabels and the merged double label(yellow indicating co-localization) for the following pairs of antibodies: A1–3, rabbitpolyclonal antibodies (pAb) to NFL and mouse monoclonal antibodies (mAb) to NFM; B1–3, rabbit pAb to NFL and mouse mAb to NFH; C1–3, rabbit pAb to NFL and mouse mAb toα-internexin; D1–3, rabbit pAb to NFM and mouse mAb to α-internexin; E1–3, rabbit pAbto NFH and mouse mAb to α-internexin; F1–3, rabbit pAb to NFL and mouse mAb toperipherin; G1–3, rabbit pAb to α-internexin and mouse mAb to peripherin; H1–3, rabbitpAb to NFM and mouse mAb to peripherin; I1–3, rabbit pAb to NFH and mouse mAb toperipherin. Scale bar, 20 μm.

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Figure 7. Co-assembly of peripherin with NF triplet proteins into single filament networkSW13vim(−) cells were quadruple-transfected with constructs that expressed peripherin,NFL, NFM and NFH and were immunostained with pairs of antibodies. Each set of 3 panelsacross represents the single immunolabels and the merged double label (yellow indicatingco-localization) for the following pairs of antibodies: A1–3, rabbit polyclonal antibodies(pAb) to NFL and mouse monoclonal antibodies (mAb) to NFM; B1–3, rabbit pAb to NFLand mouse mAb to NFH; C1–3, rabbit pAb to NFL and mouse mAb to peripherin; D1–3,rabbit pAb to NFM and mouse mAb to peripherin; E1–3, rabbit pAb to NFH and mousemAb to peripherin. Scale bar, 20 μm.

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Figure 8. Dependence of peripherin on neurofilament subunit NFLProtein extracts prepared from sciatic nerves of wild-type and LKO mice were separated onSDS polyacrylamide gel and transferred to nitrocellulose membrane. Membranes wereprobed with anti-NFL, anti-peripherin, anti-NFM and anti-NFH antibodies (A). Note thatperipherin, NFH and NFM was reduced to 28.1%, 14% and 4.7% in the absence of NFLcompared to those in wild-type mice (B). Rarity of neurofilaments was confirmed byelectron microscopy in the sciatic axons of LKO mice compared with wild-type controlswhose neurofilaments outnumber microtubules by as much as 10:1 (C, D). Neurofilamentsand microtubules are traced by blue and red colors, respectively (C1, D1). Arrows point tomicrotubules while arrowheads to neurofilaments. Scale bar, 100nm. Per, peripherin.

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Figure 9. Higher levels of peripherin immunoreactivity in motor and sensory cell bodies in micedeleted for NFLPeripherin was detected with anti-peripherin antibody in motor or sensory cell bodies inparaffin-embedded spinal cord (A, B) or dorsal root ganglion sections (C, D) from normalmice (A, C) or mice deleted for NFL (B, D). Scale bars, 10 μm and 20 μm in A, B and C,D, respectively.

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