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The Journal of Neuroscience March 1986, 6(3): 650-660

Expression of Neurofilament Subunits in of the Central and Peripheral : An lmmunohistochemical Study with Monoclonal

J. Q. Trojanowski, N. Walkenstein, and V. M.-Y. Lee The Division of , Department of Pathology and Laboratory Medicine, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

The extent to which all neurofilament (NF) subunits (NF68, 1983;Shaw et al., 1981). For example, Sharp et al. (1982) were NF150, NF200) are expressed by different populations of ma- able to distinguishtwo classesof neuronsin PNS gangliabased ture CNS and PNS neurons is controversial. We addressed this on the presenceor absenceof NF . issue in immunohistochemical studies of mature bovine tissues The absenceof one or more NF subunits in some neurons using monoclonal antibodies specific for each bovine NF sub- would necessitatedifferent hypothesesconcerning the structure unit. and function of NFs. Alternatively, microheterogeneityamong All three NF subunits were detected in the perikarya and NF proteins due to the stateof thesepolypep- neurites of both CNS and PNS neurons; they were seen in near- tides, or as a consequenceof unknown mechanisms,may ac- ly all PNS neuronal perikarya, and in all identifiable CNS and count for the apparent variable expressionof theseproteins in PNS . Most, but not all, CNS neuronal perikarya con- neurons(Goldstein et al., 1983;Nixon et al., 1982;Sternberger tained each of these NF antigens. CNS neurons devoid of im- and Sternberger, 1983).Other explanations,such as limitations munodetectable NF antigens were generally small. The pres- in the methods used to identify NF subunits in situ, are also ence of low levels of NF antigens in neurons with scant perikaryal possible(Hickey et al., 1983). may account for the apparent absence of NF immu- Immunohistochemical studiesof the in situ distribution of noreactivity in some classes of neurons, although other expla- NF subunitshave appeared(Dahl, 1983;Goldstein et al., 1983; nations, such as microheterogeneity among NF proteins, could Gown and Vogel, 1984; Hickey et al., 1983; Ostermann et al., account for this finding. 1983; Sharp et al., 1982; Shaw et al., 1981; Sternbergerand NF antigens were also seen in some cells of the diffuse neu- Sternberger, 1983; Yen and Fields, 1981). However, only lim- roendocrine system (adrenal chromaffin cells and cells of the ited populations of CNS or PNS neurons were sampled, and pars distalis and pars intermedia), but not in other types. cells of the diffuse neuroendocrine system, which share some We suggest that the expression of all three NF subunits is a properties of neurons (Marangos et al., 1982; Nolan et al., common feature of CNS and PNS neurons and their processes, 1985), were incompletely studied. Further, antibodies specific and of some cells of the diffuse neuroendocrine system. These for each of the three NF subunits rarely were used in such findings have implications for hypotheses concerning the struc- studies, many of which were conducted using indirect immu- ture and function of the intermediate filaments of neurons, and nofluorescencerather than more sensitive immunoperoxidase for hypotheses concerning neurodegenerative diseases involving methods (Hsu et al., 1981; Sternberger, 1979).Finally, marked NF proteins. immunochemicaldifferences among NF polypeptidesfrom dif- ferent vertebrateswere reported recently (Shawand Weber, 1984; The structure and function of intermediate filaments are in- Shawet al., 1984), and thesedifferences were often not consid- completely understood. Neurofilaments (NFs) are composed of ered in earlier immunohistochemical studiesof NF subunits. three subunits with apparent molecular weights of 200,000 Any one of the factors mentioned above could account for the (NF200), 150,000 (NF150) and 68,000 (NF68) Da (Liem et al., apparent absenceof one or more NF subunitsin different pop- 1978; Schlaepfer and Freeman, 1978). They are separate gene ulations of CNS and PNS neurons. products (Csoznek et al., 1980) and are immunochemically dis- In view of theseconsiderations, we probed the distribution tinct (Lee et al., 1982b). Although NF200, NF150, and NF68 of each of the NF triplet proteins in a wide variety of bovine have been localized to lo-nm-diameter filaments in neurons tissuesusing immunoperoxidasemethods and monoclonal an- (Hirokawa et al., 1984; Schlaepfer et al., 1981; Sharp et al., tibodies specificfor bovine NF antigens. 1982; Willard and Simon, 198 l), some neurons or portions of some neurons appear to lack one or more NF subunits (Dahl, Materials and Methods Monoclonal antibodies specljic for NF triplet proteins Received Feb. 19, 1985; revised May 3, 1985; acceptedJune 14, 1985. Sixteen fusionswith mousemyeloma cells (SP2/0-AG14) and spleno- This work was supported in part by NIH Grants CA-36245 (J.Q.T.) and NS- cytes from Lewis rats immunized with bovine immunogens have yielded I86 I6 (V.M.-Y.L.). J.Q.T. is the recipient of Teacher Investigator Development a library of over 200 monoclonal antibodies specific for NF subunits. Award K07 NS-00762. Appreciation is expressed to Drs. M. J. Carden and W A number of monoclonal antibodies from this library, together with the W. Schlaepferfor helpful critical comments,and to Ms. M. Obrocka for invaluable help. specifics of the fusions and characterization studies, have been Correspondenceshould be addressed to Dr. Trojanowski at Division of Neu- described (Lee et al., 1982b, 1984). The other anti-NF monoclonal ropathology, Department of Pathology and Laboratory of Medicine, University antibodieshave been characterized in a similarmanner. The specificities of Pennsylvania School of Medicine, Johnson Pavilion G2, Philadelphia, PA of thesemonoclonal antibodies for bovine NF proteinswere deter- 19104. mined as follows: (1) enzyme-linked immunosorbant assay using pur- Copyright 0 1986 Society for Neuroscience 0270-6474/86/030650-l 1$02.00/O ified bovine NF proteins; (2) immunoblot studies on cytoskeletal ex-

650 The Journal of Neuroscience Neurofilament Subunits in CNS and PNS Neurons 651

Table 1. Monoclonal antibodies to NF polypeptides and GF Table 2. Immunohistochemical results

Monoclonal Polypeptide Phosphorylation state GF antibody specificity and subunit topography Tissue NF68 NF150 NF200 protein SE3 (SE1.2B12) NF68 Noncore Neural tissues OC85 (OC3.2ES) NF150 Peripheral, nonphosphorylated *Carotid body P, N P, N P, N A TA54 (2.4FlO) NFl50 Core Cortex, cerbellar P, N P, N P, N G TA34 (2.2B12) NF150 Peripheral, phosphorylated Cortex, frontal P, N P, N P, N G TA50 (l.lC7) NF200 Peripheral, phosphorylated Cortex, occipital P, N P, N P, N G TA52 (2.2F3) NF150 + Peripheral, phosphorylated Ganglia, enteric P, N P, N P, N G NF200 Ganglia, sympathetic P, N P, N P, N A TA5 1 (4.3F9) NF150 + Peripheral, phosphorylated Ganglia, trigeminal P, N P, N P, N A NF200 Hypothalamus P, N P, N P, N G 2.2BlO GF protein Undefined Optic nerve N N N G The antibody code is in the left column, and its polypeptide specificity is indicated *Pineal gland P, N P, N P, N G in the middle column. The right-hand column contains data on the topographical P, N P, N P, N G location and phosphorylation state ofeach epitope recognized by these monoclonal Spinal cord antibodies except for 2.2BlO. The location of the NF150 and NF200 epitopes P, N P, N P, N G recognized by these antibodies in the peripheral (C-terminal or tail-piece) or core Endocrine tissues (alpha-helical) domains of these subunits is indicated (“peripheral” or “core,” respectively). If phosphate residues are present in or near these epitopes such that Adrenal gland their immunoreactivity is affected by enzymatic dephosphorylation, this fact is Cortex N N N A also noted. The NF68 epitope is unaffected by enzymatic dephosphorylation; it resides in the non-alpha-helical (“noncore”) region of NF68. See Carden et al. *Medulla P, N P, N, 0 P, N A (1985) for further details. *Pancreas N P, N P, N A *Parathyroid gland N N N A tracts of bovine spinal cord containing NF triplet proteins, glial (GF), and filament proteins, , , and other proteins; (3) Pituitary gland studies on acetone-fixed, frozen sections of bovine *Pars distalis N, 0 N, 0 N, 0 A cerebellum; and (4) immunoperoxidase studies on paraffin sections of Pars intermedia N, 0 N, 0 N, 0 A bovine cerebellum fixed by a number of different fixatives. Pars nervosa N N N G Recently, the NF epitopes recognized by these monoclonal antibodies *Submandibular gland N N N A were further defined with respect to their location in peripheral (C- terminal or tail-piece) versus core (alpha-helical) domains of NF150 *Thyroid gland N N N A and NF200, and core versus noncore regions of NF68 (see Carden et Other tissues al., 1985, for details). Further, the effect of the phosphorylation state of NF150 and NF200 on the detection of these subunits with our anti- Blood vessels A A A A bodies has also been determined (Carden et al., 1985). *Intestinal mucosa N N 0 N A Of over 200 anti-NF protein monoclonal antibodies that we could Kidney N N N A select, seven met the following criteria for use in this study: (1) They Lens A A A A are specific for only one or two NF subunits; (2) they detect NF antigens by immunoperoxidase methods; (3) they do not cross-react with fibro- Liver A A A A blast, muscle, or epithelial (i.e., vimentin, , or ) antigens *Lung N N N A using these techniques; (4) they detect NF epitopes both in perikarya Lymph node N N N A and processes of neurons. The latter point is critical since NF subunits Muscle N N N A contain epitopes that are preferentially expressed in different regions Skin N N N A (cell body or processes) of a (Goldstein et al., 1983). The mono- clonal antibodies used in this study are listed in Table 1. Spleen N N N A Testis N N N A Preparation of bovine tissuesamples for Thymus N N N A The nresence or absence of the three NF subunits and GF protein in each tissue Previous studies showed that bovine NF antigens are optimally de- is indicated as follows: A = absence of immunoreactivity; G = GF-positive cells tectable in paraffin sections following fixation of the tissues in Bouin’s ( in the CNS, emetic in the PNS); N = NF-positive neurites (axons fixative or in a mixture of mercuric chloride and formaldehyde (Hickey and ); P = NF-positive neuronal perikarya; 0 = NF-positive cells other et al., 1983). Accordingly, tissue fixed in Bouin’s solution or a mixture than classical neurons or glia (in adrenal medulla, 0 = chromaffin cells; in intestinal of 4% mercuric chloride and 8% formaldehyde was used here. All tissue mucosa, 0 = enterochromallin cells; in pars intermedia and pars distalis, 0 = cell samples were trimmed fresh to 4-5 mm thick slabs and immersion- bodies). An asterisk (*) identifies components of the diffuse neuroendocrine system fixed for 4-6 hr prior to paraffin embedding. The CNS and PNS tissues (Matangos et al., 1982; Nolan et al., 1985). and other regions examined in this study are listed in Table 2. To reveal potentially masked antigen determinants, selected mono- Immunohistochemicalprocedures clonal antibodies were applied to sections pretreated for 30 min with The immunoperoxidase methods used here have been described (Tro- one of the following: 0.1% SDS, 0.5% Triton X-100, or 0.1% trypsin, janowski et al., 1984a). In brief, the monoclonal antibodies were applied as detailed elsewhere (Troianowski et al., 1984a). Additional immu- to 6-pm-thick deparafhnized tissue sections as spent supematants fol- nohistochemical studies were conducted on selected sections using gold- lowing quenching of endogenous peroxidase activity with methanol and labeled antibodies. Briefly, colloidal gold- (40 nm gold granules) labeled hydrogen peroxide. Bound antibody was visualized with the peroxidase rabbit anti-rat IgG (Janssen Pharmaceutics, Belgium) was applied un- antiperoxidase (PAP) method (Stemberger, 1979) or the avidin-biotin diluted or diluted (1:5 with 0.5 M Tris buffer, pH 7.4, with 2% fetal method (Hsu et al., 198 1). The linking antibody used in the PAP tech- calf serum) for 1 hr to tissue sections previously incubated with hybri- nique was purchased from Cappel Labs (Cochranville, PA), and the doma supematant as in the immunopcroxidase studies. PAP reagent was obtained from Stembcrger-Meyer (Jarretsville, MD). Following the immunohistochemical procedures, all of the sections The avidin-biotin procedure was conducted according to the recom- were washed, dehydrated, coverslipped, and examined with or without mendations of the manufacturer (Vector Labs, Burlingame, CA). a counterstain. 652 Trojanowski et al. Vol. 6, No. 3, Mar. 1986

Controls and data evaluation fibrous astocytes; protoplasmic astrocytes were weakly and in- Spent supernatant from unfused mouse myeloma cells was used in par- frequently immunostained (Fig. ID). In the optic nerve (Fig. allel with the monoclonal antibodies to monitor nonspecific . 2B) and pineal gland (Fig. 20), GF protein-positive astrocytes In addition, a monoclonal antibody specific for bovine GF protein (Lee were numerous.No GF immunoreactivity was seenin neurons, et al., 1984) was used in parallel with the other antibodies as a control. ependymal or oligodendroglial cells, , blood vessels,etc. GF protein, initially isolated from gliotic brain (Eng et al., 197 l), is the major protein of the intermediate filaments of astrocytes; it is also Immunoreactive NF subunitsin the PNS present in enteric glia and a few other cell types (see review by Eng and Consecutive sectionsof the carotid artery bifurcation and ca- DeArmond, 1983). Since axons were nearly ubiquitous in the tissues assayed, they pro- rotid body, paravertebral sympathetic ganglia, and trigeminal vided endogenous positive controls for the anti-NF monoclonal anti- gangliawere immunostained with the anti-NF monoclonal an- bodies. A section of bovine cerebellum was included in each experiment tibodies. In addition, enteric ganglia of the small and large in- to provide a positive control for the monoclonal antibody to GF protein. testinesand a number of other microscopicganglia lying within Only experiments in which all of these controls were successfully exe- or adjacent to other tissues,such as the submandibulargland, cuted underwent further evaluation for inclusion in this study. pancreas,etc., were similarly immunostained. In each of these The immunohistochemical results were evaluated as in a previous tissues, NF-positive neurites were prominent. All identified study (Hickey et al., 1983). The presence or absence of NF68, NFl 50, clusters of axons were immunostained by each NF subunit NF200, and GF immunoreactivity in consecutive sections from each specific antibody. No clusters failed to expressall of the tissue was tabulated. NF triplet proteins. This was most evident within and around ganglia (Figs. 3, A-C, 4, A-C, 5, A, D), where densely packed Results immunostained axons could be compared in adjacent sections. Neuronal perikarya in these ganglia exhibited intense im- Generalobservations munoreactivity for eachof the NF subunits(Figs. 3, A-C, 4, A- The sensitivity of the immunoperoxidase methods exceeded C, 5, A and U). In contrast with the findingsin the CNS, nearly that of the immunogold method. The results in Bouin’s and every PNS neuron exhibited immunoreactivity for each of the mercuric chloride/formaldehyde-fixed tissues were similar. NF NF subunits; negative neuronswere exceptional (compareFig. and GF immunoreactivity in neurons and glia, respectively, was 1, A-C with Fig. 3, A-C). Although formal quantitation wasnot always diffuse; no aggregatesof NF immunoreactivity, similar undertaken, fewer than 1% of identified PNS neurons lacked to those in transformed cells (Lee, 1985; Lee and Page, 1984; NF protein immunoreactivity, while asmany as 30% of neurons Lee et al., 1982a; Trojanowski et al., 1984b) were seen.The in a given CNS region lacked NF immunoreactivity. However, resultsobtained usingthe PAP and avidin-biotin methodswere the PNS neurons were uniformly large, with abundant cyto- equivalent, and treatment of the sectionswith Triton, trypsin, plasm(Figs. 3-Q whereaspopulations of smallerneurons were or SDS did not improve the results. Except for the fact that frequent in the CNS. TA54 weakly stained neurons and axons, possibly becauseof One ofthe anti-NF150 monoclonal antibodies(OC85) yielded the core location of the epitope it recognizes,no simple corre- a pattern of immunoreactivity in the intestine that differed from lation between the expression of an NF epitope and its domain all of the others. It stained neuronal perikarya and neurites, as or phosphorylation statuswas evident. The immunoperoxidase did the other anti-NF monoclonal antibodies, but in addition resultsare consideredin further detail below (seealso Table 2 it stainedscattered enterochromaffin cells (Marangos et al., 1982; and Figs. l-6). Nolan et al., 1985; Weiss, 1983) of the intestine (Fig. SC’).The failure of any of the other monoclonal antibodies to immuno- stain thesecells and the failure of this antibody to stain other Immunoreactive NF subunitsin the CNS cells in endocrine tissues(e.g., submandibular, pancreas,para- Consecutivesections of frontal, occipital, and cerebellar cortex thyroid, or thyroid glands)suggests that OC85 cross-reactswith were examined in addition to consecutive sections of hypo- an antigen specificto enterochromaffin cells. Dilution of OC85 thalamus,optic nerve, pineal gland, retina, and spinal cord. In 1:50 with Tris buffer eliminated all neuronal staining, but it still each region, neurites (axons and dendrites) consistently con- stainedenterochromaffin cells intensely (Fig. 5C). tained each of the NF subunits. In subcortical (Fig. NF immunoreactivity in the carotid body differed from that 1, A-C), optic nerve (Fig. 2A) and spinal cord, where it was observed in PNS ganglia; only a few cells were stained possible to survey larger numbers of axons, all identifiable axons with the NF subunit-specific monoclonals (Fig. 6C). Some of expressed NF68, NFl50, and NF200 immunoreactivity. In areas these NF-positive neurons were also seen outside the carotid enriched in neuron cell bodies, such as cortex, hypothalamus, body in the wall of the adjacent carotid artery (Fig. 60). Axons spinal cord, or retina, large numbers of neuronal perikarya were in the vicinity of the carotid body were uniformly positive with immunostained with each of the NF subunit specific mono- each of the anti-NF monoclonal antibodies (Fig. 6C’). On the clonal antibodies. However, neurons with low levels of or no other hand, none of the smaller cells within the carotid body detectable immunoreactivity, regardlessof the subunit specific was similarly immunostained. Muscle cells and fibroblasts in monoclonal antibody used,were frequently seen(Fig. 1, A-C). the carotid artery were also negative. No blood vesselsother Thesewere generallysmaller neurons with scantcytoplasm, such than the carotid artery contained similar NF-positive neurons as thosein granular layers of cerebral cortex or the granular cell within their walls. neuronsof cerebellarcortex. In the pineal gland (Fig. 2C), only Other than the neurons and neuronal processesdescribed rare cell bodies with NF immunoreactivity were seen(a maxi- above, no other elements in the tissuesdescribed here were mum of 3-5 per horizontal section through the entire pineal). immunostained with theseanti-NF monoclonal antibodies. Immunostained axons were similarly infrequent in the pineal In consecutive control sectionsthrough the samePNS tissues, gland. The scarcity of neurons and axons in the pineal is con- only glial cells in enteric gangliaof the intestine expressedGF sistent with current views on the histological composition of protein (Fig. 5B). Rarely, an occasional satellite cell was im- this gland(Weiss, 1983). No other elementsin any of theseCNS munostainedwith the monoclonal antibody to GF protein (Fig. regions(i.e., blood vessels,glial cells, leptomeninges,etc.) con- 40). Consistentwith earlier immunoperoxidasestudies of nor- tained NF immunoreactivity. mal and degenerating rat and human peripheral nerve, we did In adjacent control sections from these same tissues, the not detect GF immunoreactivity in Schwanncells (Trojanowski monoclonal antibody to GF protein intensely stainednumerous et al., 1984b). Whether Schwann cells expressthe same GF The Journal of Neuroscience Neurofilament Subunits in CNS and PNS Neurons 653

Figure I. Consecutive sections through layers III-VI and the subcortical white matter of occipital cortex immunostained with monoclonal antibodies to NF68 (SE3 in A), NF150 (X85 in B), NF200 (TA50 in C), and GF protein (2.2BlO in D). Most neuronal perikarya express each of the NF subunits; NF-negative neurons are predominantly small and located in the granular layer of cortex. GF immunoreactivity is restricted almost exclusively to fibrous astrocytes; protoplasmic astrocytes are infrequently immunostained. AI1 of the sections in Figures l-6 were counterstained with hematoxylin and enlarged x 250, except Figure 5B, which is enlarged x 500. protein present in astrocytes remains unresolved (Dahl et al., monoclonal antibodies to NF subunits. A variety of other tissues 1982; Eng and DeArmond, 1982; Schlaepfer et al., 1979). were also examined in the same manner, including large blood vessels (both arteries and veins), kidney, lens, liver, lung, lymph Immunoreactive NF subunits in endocrine and nodes, skin, smooth and striated muscle, spleen, testis, and thy- other tissues mus. Consecutive sections through endocrine tissues, including the The pattern of NF immunoreactivity in the adrenal and pi- adrenal gland, pancreas, parathyroid gland, pituitary gland, sub- tuitary glands differed from that seen in the other tissues dis- mandibular gland, and thyroid gland, were examined with the cussed in this section. A dense plexus of neurites in the adrenal 654 Trojanowski et al. Vol. 6, No. 3, Mar. 1986

Figure 2. Consecutive transverse sections through optic nerve (A, B) and pineal gland (C, D) immunostained with a monoclonal antibody specific for NF68 (SE3 in A) or NFl50 and NF200 (TA5 1 in C) or for GF protein (2.2B 10 in B and D). All optic nerve axons contain NF immunoreactivity; astrocytes are GF-positive. In the pineal gland, NF-positive neurons and neurites are rare; numerous astrocytes with immunoreactive GF are seen.

medulla (Fig. 64 wasimmunostained with eachof the anti-NF axons that were positively stained with monoclonal antibodies antibodies, as were occasionalganglion cells (one to five per specific for each of the NF subunits; a few of these axons ex- section of adrenal medulla). Chromaffin cells were negative in tended into the pars intermedia and pars nervosa (Fig. 6B). sectionsstained with the antibodiesspecific for NF68 or NF200, Aggregatesof NF protein immunoreactivity, which appearedto but those specific for NF150 or for NF150 and NF200 stained be in the extracellular spaceof the parsnervosa, werealso noted. numerousadrenal chromaffin cells. Adrenal cortical cells were In addition, rare cell bodiesscattered in the parsintermedia and negative, but NF-positive axons in large nerves of the cortex pars distalis near the pars nervosa were immunostained with were observed. the monoclonal antibodies specific for NF68, NF150, and In the pituitary gland, the pars nervosa contained numerous NF200; none were present in the pars nervosa (Fig. 6B). The Journal of Neuroscience Neurofilament Subunits in CNS and PNS Neurons 655

Figure 3. Consecutivesections through trigeminal ganglion immunostained with monoclonalantibodies to NF68 (SE3in A), NF150 (TA34 in B), NF200(TA50 in C’),or GF (2.2BlOin D). All neuronsare positive for eachNF subunit;no GF-positiveglial cellsare seen.

The parenchyma of the other organsdiscussed in this section chyma (seeFig. 5, A and D). Occasional ganglia comprised of was devoid of any NF immunoreactivity except for that seen 10-20 neuronscontaining immunoreactive NF68, NF150, and in nerves traversing eachorgan. The walls of arteries and veins NF200 were seenat the periphery of some of the endocrine were devoid of immunostained axons or cell bodies except in tissues(Fig. 5D). the vicinity of the carotid artery, as described above. Axons Aside from the cells and processesdescribed above, no other with immunoreactive NF68, NF150, and NF200 were seenin NF immunoreactivity wasseen in any of the other components sectionsfrom each of these organs, but they were not as nu- of the tissueslisted in Table 2. merous as those seenin the adrenal or pituitary glands (see In consecutive control sectionsof the sametissues considered below). Immunostained nerves were relatively rare (O-10 nerve here, only occasionalastrocytes in the pars nervosa were GF fasciclesper section)in thesetissues; they were most prominent positive. Although Hatfield et al. (1984) have recently dem- in the capsuleof these organsand lessfrequent in the paren- onstrated GF protein in the epithelium of mouse lens using 656 Trojanowski et al. Vol. 6, No. 3, Mar. 1986

Figure 4. Consecutivesections through an autonomicganglion immunostained with monoclonalantibodies to NF68 (SE3in A), NFl50 (TA34 in B), NF200(TA50 in C), andGF protein(2.2BlO in D). All neuronsare positive for eachNF subunit.No &al cellscontain GF proteinwith the exceptionof a raresatellite cell (arrowhead). antiserum raisedto human GF antigens,bovine lens was neg- ative when stainedwith our monoclonal antibody to bovine GF Discussion protein. These studiesdemonstrate the ubiquitous distribution of each Although the monoclonal antibody to bovine GF protein was of the three NF subunitsin diverse populationsof PNS neuronal used as a control antibody in thesestudies, it is of interest to perikarya and in diverse populations of PNS and CNS axons note that, with the few exceptions pointed out earlier, the GF using well-characterized monoclonal antibodies and immuno- protein epitope recognized by this monoclonal antibody has a histochemistry. The majority of CNS neuronal perikarya also distribution similar to that observed in other immunohisto- contained each of these antigens.This has important implica- chemical studies using anti-GF protein antiserum (Eng and tions for hypotheses concerning the structure and function of DeArmond, 1982). the intermediate filaments of neurons. The Journal of Neuroscience Neurofilament Subunits in CNS and PNS Neurons 657

Figure 5. Sections through intestine (A-C) and submandibular gland (0) immunostained with monoclonal antibodies to NF200 (TA50 in A and D), a monoclonal antibody (OC85) specific for NF150 (C), and the anti-GF monoclonal antibody (2.2BlO in B). All of the neurons and axons in A and Dare NF-positive. Enteric glia (B, arrowhead) express GF protein. OC85 cross-reacts with enterochromaffin cell-specific antigens (0). Diluted 1:50 here, OC85 does not immunostain neurons and axons, while enterochromaffin cells are still positive. OC85 was the only anti-NF monoclonal antibody in this study that stained enterochromaffin cells.

Our data support the generality of current modelsof NF struc- lian NF proteins are insoluble on extraction in Triton X-100 ture, which indicate that the intermediate filaments of neurons (Liem et al., 1978; Schlaepfer and Freeman, 1978) and that are comprised of a triplet (NF68, NF150, NF200) of polypep- these Triton-insoluble extracts contain lo-nm-diameter fila- tides (Liem et al., 1978; Schlaepfer and Freeman, 1978). The ments that can be decorated by antibodies specific for each of immunohistochemical demonstration of NF68, NF150, and thesesubunits (Schlaepfer et al., 1981; Sharpet al., 1982,Willard NF200 antigensin neuronsmeans that thesesubunits are avail- and Simon, 1981). This suggeststhat the monoclonalantibodies able for incorporation into NFs, but it doesnot prove they are usedin these studiesprobably bind to NF subunits assembled assembledinto filaments. However, it is known that mamma- into filaments. Our data are also important for evaluating hy- 658 Trojanowski et al. Vol. 6, No. 3, Mar. 1986

Figure 6. Sections of adrenal medulla (A), pituitary gland (B), carotid body (c), and adjacent carotid artery (D) immunostained with a monoclonal antibody specific for both NF150 and NF200 (TA5 1). In the adrenal medulla many labeled neurites and a labeled ganglion cell are seen. Chromalhn cells contain NF immunoreactivity not apparent at this magnification. In the pars nervosa (B, upper halfi axons are NF-positive; some NF immunoreactivity is in aggregates that may be extra-axonal (large arrowhead). A few cells in the pars intermedia also contain NF immunoreactivity (small arrowheads). In the carotid body, neurons and axons contain NF immunoreactivity. NF-positive neurons are seen also in the adjacent wall of the carotid artery (0). No other blood vessels contained similar NF-positive cells. x 250. potheses concerning the function of mammalian NFs. The near- of the monoclonal antibodies used in this study for bovine NF ubiquitous presenceof each of the NF polypeptides in mature proteins have been rigorously evaluated (Carden et al., 1985; CNS and PNS neuronsand their processes,coupled with their Hickey et al., 1983; Lee et al., 1982b, 1984), immunohisto- absencein other mature cell types, suggeststhat NFs subserve chemistry alone cannot provide conclusive proof that the an- someunique neuronal function. tigensdetected in situ with theseantibodies are indeed NF poly- The data presentedhere must be viewed with the limitations peptides.It is known that antibodies may recognize epitopesin of immunohistochemistry in mind. Although the specificities unrelated proteins (Dulbecco et al., 1981), in addition to the The Journal of Neuroscience NeurofilamentSubunits in CNS and PNS Neurons 659 immunogensagainst which they are raised. Monoclonal anti- inantly small, with scant cytoplasm, the absenceof NF im- bodies that detect epitopes shared by more than one classof munoreactivity may be a false-negativeresult due to low levels intermediatefilaments have been described(Lee and Page, 1984; of NF antigens.Since all identified axons expressedNF antigens, Lee et al., 1984; Prusset al., 1981) and antiserum to a synthetic it is possiblethat NF subunitsare rapidly exported to neuronal peptide has been shown to cross-reactwith NF150 in mice and processesfollowing synthesisand that only small amounts of guineapigs (Drager et al., 1983; Trojanowski et al., 1985). Fur- NF antigens remain in the perikaryon of someneurons. Alter- ther, possibly becauseNF subunits may undergo posttransla- natively, the functional state of the cell, at the time of fixation tional modifications, or becauseof other yet-to-be-defined fac- (mediated by yet to be defined processes),could modify the tors that affect the immunoreactivity of NF subunits, anti-NF epitopesdetected by our monoclonal antibodiesin selectedneu- monoclonal antibodiesmay detect NF proteins in axons but fail rons. Finally, it is possiblethat some neuronsindeed lack NF to recognize them in neuronal perikarya, where they are syn- subunits. However, we speculatethat this is a lesslikely pos- thesized (Gown and Vogel, 1984; Hickey et al., 1983; Stern- sibility than the others mentioned above. berger and Sternberger, 1983). With these cautions in mind, we feel that our data suggest Conclusive proof that the antigens stained here are indeed that the expressionof all three NF subunits is a fundamental NF subunits would require their isolation from the immuno- property of CNS and PNS neuronsand their processes.These stainedcells. NF proteins have been isolated from someof the observations are important for the formulation of hypotheses tissuesstudied here, e.g., from peripheral nerve and spinal cord concerningthe structure and function of NF proteins. They also (Carden et al., 1985; Liem et al., 1978; Schlaepferet al., 1979), have implications for hypothesesconcerning the etiology of neu- and retina, optic nerve, and pars nervosa (Trojanowski et al., rodegenerativediseases that putatively affect the intermediate 1985).However, sincenerves are ubiquitous, attemptsto extract filaments of neurons(Autilio-Gambetti et al., 1983; Schlaepfer, NF proteins from distinct populations of cells in the tissues 1983). examined here would result in the contamination of such ex- Note added in proof: Our findings here with the monoclonal tracts by the NF proteins in peripheral nerves. The isolation of antibody 2.2BlO raised to GF protein are similar to NF proteinsfrom cellsgrown in primary culture is possible.For thosereported by Jessenet al. [(1984) J. Neurocytol. 13: 187- example, NF proteins have been isolatedfrom cultured adrenal 200] for another monoclonal antibody (anti-GFAP-3) raisedto chromaffin cells (Bader et al., 1984) and sympathetic neurons astrocyte GF protein. The epitope recognizedby thesetwo an- (Lee, 1985). However, this strategy also has limitations. For tibodies is expressedby GF protein of astrocytes and enteric example, different tissue culture conditions dramatically affect glia. the expressionof NF and GF proteins in cultured cells (Lee, 1985; Lee and Page, 1984; Raff et al., 1983). This may explain References why we detected only NF 150 in bovine adrenal chromaffin cells Autilio-Gambetti,L., P. Gambetti,and R. Crane (1983) Pairedhelical in situ, whereasBader et al. (1984) detected all three NF proteins filaments:Relatedness to neurofilamentsshown by silverstaining and in primary cultures of bovine adrenal chromaffin cells. reactivity with monoclonalantibodies. In Biological Aspects of Alz- In order to overcome the limitations inherent in immuno- heimer’s Disease, R. Katzman, ed., pp. 117-124,Banbury Report histochemistry, we utilized a panel of monoclonal antibodies Series,Cold SpringHarbor Laboratory,NY. with exhaustively characterized specificitiesfor different bovine Bader,M. F., E. Georges,W. E. Mushynski,and J. M. Trifaro (1984) Neurofilamentproteins in culturedchromaffin cells. J. Neurochem. proteins and applied them to a wide range 43: 1180-l 193. of tissuesfixed by different methods. We also employed the Carden,M. J., W. W. SchIaepfer,and V. M.-Y. Lee (1985) The struc- most sensitivecurrently available immunoperoxidasemethods ture, biochemicalproperties and immunogenicityof neurofilament (Hsu et al., 1981; Stemberger, 1979). Finally, we usedmono- peripheralregions are determined by phosphorylation.J. Biol. Chem. clonal antibodies raised against immunogens from the same 260: 9805-9817. speciesin which the immunohistochemical studies were con- Czosnek,H., D. Soifer,and H. M. Wisniewski(1980) Studieson the ducted. This is of critical importance in view of the marked biosynthesisof neurofilamentproteins. J. Cell Biol. 85: 726-734. immunochemical differencesin NF proteins of even closely re- Dahl, D. (1983) Immunohistochemicaldifferences between neurofil- lated species(Shaw and Weber, 1984; Shaw et al., 1984). For amentsin perikarya,dendrites and axons.Exp. Cell Res.149: 397- 408. example, in our library of anti-NF150 monoclonal antibodies, Dahl, D., N. H. Chi, L. E. Miles,B. T. Nguyen,and A. Bignami(1982) only thosethat seecore (in contrast to peripheral) NF150 epi- Glial fibrillary acidic(GFA) proteinin Schwanncells: Fact or artifact. topes recognize rat NF150 (Lee et al., unpublished observa- J. Histochem.Cytochem. 30: 9 12-918. tions). Thesefactors alonecould account for the failure to detect Drager,U. C., D. L. Edwards,and J. Kleinschmidt(1983) Neurofila- NF subunitsin someneurons if the antibodies usedto localize mentscontain a-melanocyte-stimulating hormone (a-MSH)-like im- theseantigens in a given specieswere raised and characterized munoreactivity.Proc. Natl. Acad. Sci. USA 80: 6408-6412. usingNF proteins from another species. Dulbecco,R., M. Unger, M. Bologna,H. Battifora, P. Syka, and S. Despitethe exhaustive characterization of thesemonoclonal Okada (1981) Cross-reactivitybetween Thy- 1 and a componentof intermediatefilaments demonstrated using a monoclonalantibody. antibodies, a novel cross-reactionwas observed in gut; one of Nature292: 772-774. the anti-NF150 monoclonal antibodies (OCSS) cross-reacted Eng,L. F., J. J. Vanderhagen,A. Bignami,and B. Gerstl (1971) An with an antigenrestricted to enterochromaffin cells. This antigen acidic proteinisolated from fibrousastrocytes. Brain Res.28: 351- is unlikely to be neuron-specificenolase or chromogranin, both 354. of which are widely distributed throughout the diffuse neuroen- Eng,L. F., andS. J. DeArmond (1983) Immunochemistryof the &al docrine system (Marangos et al., 1982; Nolan et al., 1985). fibrillary acidic protein. In Progress in Neuropathology, H. M. Zim- Additional studieswill be necessaryto define the nature of this merman,ed., pp. 19-39,Raven, New York. cross-reaction.Since antiserum raised to synthetic alpha-MSH Goldstein,M. E., L. A. Stemberger,and N. H. Stemberger(1983) cross-reactswith an epitope present in NF150 (Drager et al., Microheterogeneity(“neurotypy”) of neurofilamentproteins. Proc. Natl. Acad. Sci.USA 80: 3101-3105. 1983; Trojanowski et al., 1985), it is not surprising that an anti- Gown, A. M., and A. M. Vogel (1984) Monoclonalantibodies to NF monoclonal antibody cross-reactswith antigens unrelated humanintermediate filament proteins. II. Distributionof filament to NF proteins. proteinsin normalhuman tissues. Am. J. Pathol.114: 309-321. The failure to detect bovine NF antigensin someofthe bovine Hatfield, J. S., R. P. SkoK,H. Maisel,and L. F. Eng (1984) GliaI neurons studied here is intriguing, but it must be interpreted fibrillary acidicprotein is localized in the lensepithelium. J. CellBiol. with caution. Becausethe NF-negative neuronswere predom- 98: 1895-1898. 660 Trojanowski et al. Vol. 6, No. 3, Mar. 1986

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