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Phosphorylation protects neurofilaments against proteolysis
Journal of Neuroimmunology, 14 (1987) 149-160
149
Elsevier JNI 00458
Phosphorylation protects neurofilaments against proteolysis Margi E. Goldstein *, Nancy H. Sternberger and Ludwig A. Sternberger Center for Brain Research, University of Rochester School of Medicine and Dentistry, Rochester, N Y 14642, and Departments of A natomy, Neurology and Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. (Received 1 April 1986) (Revised, received 25 August 1986) (Accepted 26 August 1986)
Key words: Neurofilament conformation; Monoclonal antibodies; Peroxidase-monoclonal antiperoxidase technique; Neurofilament protease, calcium independent
Summary During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that (1) phosphorylated neurofilaments are protected against proteolysis, and (2) dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.
* In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience. Supported by National Science Foundation grant BNS-8506474 and by National Institutes of Health grants NS 21681 (Javits Award), NS 17652, and NIMH F31 MH09100. Address for correspondence: Dr. Nancy Sternberger, Department of Anatomy, University of Maryland School of Medicine, 655 W. Baltimore Street, Ba/dmore, MD 21201, U.S.A. 0165-5728/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
150 Introduction
The finding that some monoclonal antibodies to neurofilaments react with phosphorylated epitopes has been dependent upon (a) inhibition of the antibodies by inorganic phosphate, acting as a hapten (Sternberger and Sternberger 1983), (b) failure of these antibodies to react with the nonphosphorylated 170 kDa component of the heavy neurofilament peptide (Nf-H) (Goldstein et al. 1987), and (c) failure to react with phosphatase-treated neurofilaments (Sternberger and Sternberger 1983). That the phosphatase effects, indeed, were due to phosphatase rather than any other component in the enzyme preparation was surmised from their inhibition by inorganic phosphate, an inhibitor of phosphatase. However, more recently we observed that incubation of neurofilaments with phosphatase produced a number of lower molecular weight forms in addition to those that can be attributed to dephosphorylation alone. The present paper examines these effects.
Materials and methods
Monoclonal antibodies Monoclonal antibodies were produced by the method of Ki3hler and Milstein (1976a, b) and Fazekas de St. Groth and Scheidegger (1980) as described previously (Sternberger et al. 1982). Antibodies 02-135 and 06-32 reacted with nonphosphorylated neurofilaments (anti-nPNf), antibodies 03-44, 06-17, 04-7 and 07-5 with phosphorylated neurofilaments (anti-PNf), and antibody 02-40 with a nonphosphorylated epitope in both, nonphosphorylated and phosphorylated neurofilaments.
Cytoskeletal preparation Cytoskeletal proteins were prepared by the method of Chiu et al. (1981, 1982) and Eng and DeArmond (1983) as described previously (Go!dstein et al. 1983). Protein concentrations were determined by the method of Lowry et al. (1951) or Bradford (1976).
Gel electrophoresis One-dimensional gels were set up by the method of Laemmli (1970) as described previously (Goldstein et al. 1983). The separating gels contained 7.5% acrylamide. The gels were either stained in 0.125% Coomassie blue R-250 in 50% methanol/10% acetic acid, or immunoblotted by the method of Towbin et al. (1979) as described previously (Goldstein et al. 1983) using 0.025 M Tris-HC1, pH 8.03/0.05 M NaC1/0.19 M glycine/10% methanol.
Immunocytochemistry Immunoblots were stained immunocytochemically (Sternberger et al. 1970) using mouse peroxidase-antiperoxidase complex made from monoclonal antiperoxidase (Sternberger 1986).
OF NEUROFILAMENTS.
SUMMARY OF IMMUNOBLOTS
P
200
170 150 130 LMF
Anti-PNf
(03-44, 06-17, 04-7, 07-5) -
+
Masked Masked + + + +
++++ ++++
Masked Masked ++++
+++
++++
+
+++
+
_.
_
+
_
_
+
+ +
+ +
+
+
+
++
+
++++ +++
++++
+
+
++
++++
+
++
++++
P+Ap
P
++
++++
+ +
_
+ +
++++
++
-
++
+
-
-
-
+
+
+
+
Masked Masked
++
--
Masked Masked ++++
+
.
Soluble fraction
B
.
+4
+++
+ +
-
+ +
++++
+ +
.
+
P+Pi
.
.
+
++
+
_
+ +
++
+
-
+
.
.
P+Ap
+ + + + + + .
-
B
fragment
+
+
B
+ + +
--
+
+ +
. . .
--
.
--
+
+ + + +
. .
++++
-
P
. .
+ +
--
+
4 +
+
+
P+Pi
Affinity-purified Nf-H
+ . q: + + + . . .
-
P
Chymotryptic
+ +
_
+
+ +
+
+
P+Ap
B, buffer; P, p h o s p h a t a s e ; P + Pi, p h o s p h a t a s e in p h o s p h a t a s e buffer; P + Ap, p h o s p h a t a s e plus a m i n o p h y l l i n e ; n P N f , n o n p h o s p h o r y l a t e d n e u r o f i l a m e n t s ; PNf, phosphorylated neurofilaments. a A n t i b o d i e s to n P N f epitopes m a s k e d b y p h o s p h o r y l a t i o n . b A n t i b o d y to n P N f epitopes accessible in n P N f as well as PNf. c Lower molecular weight fragments.
-
++++
+ + -
150 130 LMF
-
150 130 LMF c
++++ ++
+++
170
200 170
++++
200
Insoluble residue
B
P+Pi
T r e a t m e n t of p r o t e i n s
fraction Cytoskeletal p r o t e i n s (kDa)
Phosphorylationindependent anti-nPNf (02-40) b
(02-135, 06-32) "
Phosphorylationdependent anti-nPNf
antibodies
Monoclonal MW
E s t i m a t e s of staining are a v e r a g e s w h e r e different a n t i b o d i e s o f the s a m e g r o u p e x h i b i t e d d i f f e r e n t d e g r e e s of staining ( m i c r o h e t e r o g e n e i t y , G o l d s t e i n et al. 1983, 1987). W e a k e r staining n o n t r i p l e t n e u r o f i l a m e n t b a n d s with a p p a r e n t m o l e c u l a r w e i g h t s of 178, 164, 145 a n d 124 k D a not included.
PROTEOLYSIS AFTER DEPHOSPHORYLATION
TABLE 1
~ O n -
-- N
N
N
2
¸-¸
II
C '~
N
M
U m
Fig. 1. Immunoblots of neurofilaments degraded after dephosphorylation. Cytoskeletal preparations were incubated at 32°C for 20 h, and the proteins separated into precipitates (a and b) and supernatants (c and d). Incubation in Tris buffer (a and c, lanes 1), Tris buffer plus 250 ~ g / m l alkaline phosphatase (a and c, lanes 2), sodium phosphate buffer plus 250/~g/ml alkaline phosphatase (b and d, lanes 1), or Tris buffer plus 250 ~ g / m l alkaline phosphatase and aminophylline (b and d, lanes 2). Immunocytochemical staining with the antibodies listed. Molecular weight standards are myosin, 200 kDa; fl-galactosidase, 116.5 kDa; phosphorylase b, 92.5 kDa, bovine serum albumin, 66 kDa and ovalbumin, 45 kDa.
153
lmmunoprecipitation Neurofilaments were precipitated with anti-nPNf 02-135 and anti-PNf 04-7 by the method of Bader et al. (1984) as described in the accompanying paper (Goldstein et al. 1987). Anti-nPNf and anti-PNf were replaced with monoclonal antibody to L H R H ( K n a p p and Sternberger 1984, 1986) in control precipitations.
Chymotrypsin digestion Cytoskeletal proteins, at concentrations of 3 m g / m l in 0.1 M Tris-HC1, p H 8.0, were incubated with 1 # g / m l chymotrypsin at 32°C for 1 h. Phenylmethylsulfonyl fluoride (PMSF) was added to stop the reaction and the proteins were centrifuged at 15 600 × g in an Eppendorf microcentrifuge for 30 rain. Precipitates and supernatants were electrophoresed as such or treated with E. coli alkaline phosphatase prior to electrophoresis as described below.
Enzymatic dephosphorylation Cytoskeletal proteins, immunoprecipitated Nf-H, or chymotryptic fragments of neurofilaments were dephosphorylated with E. coli alkaline phosphatase (Worthington) as described in the accompanying paper (Goldstein et al. 1987). Phosphatase activity was inhibited by the addition of 0.2 M sodium phosphate or 2 m M aminophylline.
Results When cytoskeletal preparations were incubated in E. coli phosphatase and the ensuring precipitates immunoblotted, staining with anti-nPNf revealed N f - H as a
Fig. 2. Stability of dephosphorylated chymotryptic fragment. Cytoskeletal proteins treated with chymotrypsin and separated into precipitate (lane 1) and supernatant (lanes 2 and 3) were incubated in Tris buffer (lanes 1 and 2) or 250 #g/ml alkaline phosphatase at 32°C for 20 h (lane 3), transblotted and stained immunocytochemicallywith anti-nPNf 02-135. The molecular weight of the 160 kDa polypeptide released into the supernatant (*) became reduced to 130 kDa (**), upon treatment with phosphatase.
154 0
t M,+
~
w
g
+
b
=
+++
+P <
+++
2 1 2 ..... 2 +++++++++ ++ +++~++++++++++++++~++++ +
.... t
+
+
+ ;
++++++++++ +
+
....+ ++.... +
++++++++ + ++++++++
++ +..... +t +++++ +
.............. + ++++++++ ++.... ++++++ ++++++ ++ ++++++ .....
+ ++++
++
< +++++
+ +....
+ I +++
+++++ +++ ++ ++
+
+++ +++++ ++++++++++++++++ +
++++++++++?+++ +++++++++++?+++++ ++++++++++;+
Fig. 3. Stability of neurofilaments upon dephosphorylation after affinity purification with anti-nPNf. Cytoskeletal proteins immunoprecipitated by anti-nPNf 02-135 were incubated at 32°C for 20 h in Tris buffer (a), Tris buffer plus 250 ~ g / m l alkaline phospliatase (b), sodium phosphatase buffer plus 250 / t g / m l alkaline phosphatase (c, lanes 1), or Tris buffer plus 250 ~tg/ml alkaline phosphatase and aminophylline (c, lanes 2). The transblots were stained immunocytochemically with anti-nPNf 02-135, 06-32 and 02-40, and anti-PNf 03-44, 06-17, 04-7 and 07-5. Molecular weight standards as in Fig. 1. Dephosphorylation intensifies the 170 kDa band (**) and reveals two proteins at 145 (*) and 130 kDa (*).
155 O
-t
N
N
k
b
)
~
iiii~iiiii~ iii~i~i
'7
~i!!~!i!i!i~i ~ii
i~i~:~iii
iliii
!~
:II~'!~!!~!i ~ ii~I,!ili!~!iiiii!iiii!!~!!i!iiiii~!i! !! ~! ~i~i!!iiiii !!i !!!!~!iiiii~!!!i!!i!ii!!!!~!iiiii~i iii!~iiiiiii!!~!i!ili~i!~!!i!iiiiiii!ii!!!!ii!ii!~ ~ ~i~ ~ ~~!i~,~~ili!~i"~!i~ Fig. 4. Stability of neurofilaments after affinity purification with anti-PNf. Cytoskeletal proteins immunoprecipitated by anti-PNf 04-7 were incubated at 32°C for 20 h in Tris buffer (a), Tris buffer plus 250 # g / m l alkaline phosphatase (b), sodium phosphate buffer plus 2 5 0 / t g / m l alkaline phosphatase (c, lanes 1), or Tris buffer plus 250 / t g / m l alkaline phosphatase and aminophylline (d, lanes 2). The transblots were stained immunocytochemically with anti-nPNf 02-135, 06-32 and 02-40, and anti-PNf 03-44, 06-17, 04-7 and 07-5. Molecular weight standards as in Fig. 1. Dephosphorylation intensifies the 170 kDa band (**) and reveals two proteins at 145 (*) and 130 kDa (*).
156 broad band that included lower molecular weight forms and extended over most of the immunoblot strip (Fig. la, Table 1). Overall staining was also intensified compared to controls incubated in the absence of phosphatase. Staining with anti-PNf, on the other hand, was reduced, as expected, after incubation with phosphatase (Sternberger and Sternberger 1983), and the breakdown products could not be detected. Cytoskeletal preparations incubated in buffer did not reveal lower molecular weight breakdown products nor did breakdown occur when inhibitors of phosphatase, such as inorganic phosphate or aminophylline, were added to the phosphatase (Fig. lb). Proteolysis upon phosphatase treatment was not affected by divalent metal-chelating agents (EDTA, EGTA) nor did inhibitors of proteases, such as leupeptin, PMSF or peptastatin affect the breakdown. Cytoskeletal preparations incubated in buffer released into solution no significant amount of protein detectable by immunoblotting with anti-nPNf 02-135 and 06-32 (Fig. lc). On the other hand, incubation in phosphatase solubilized a considerable portion of the Nf-H breakdown products, as well as traces of the 170 kDa component corresponding to the dephosphorylated fraction of intact Nf-H (Julien and Mushynski 1982; Carden et al. 1985). Again, release of material into solution by phosphatase treatment was inhibited by inorganic phosphate and aminophylline (Fig. ld). o n immunoblots of chymotrypsin-digested neurofilaments, all seven antibodies stained the 160 kDa fragment released into the supernatant. None of the antibodies stained the 40 kDa chymotryptic fragment associated with the insoluble neurofilament core (Geisler et al. 1982, 1983; Weber et al. 1983). In addition to the 160 kDa chymotryptic product which is stained by all seven antibodies, anti-nPNf stained a protein band at an apparent molecular weight of 130 kDa. When the 160 kDa fragment was incubated with alkaline phosphatase, its apparent molecular weight became reduced, but no additional degradative products were stained (Fig. 2, Table 1). Incubation with E. coli alkaline phosphatase of Nf-H immunoprecipitated by anti-nPNf 02-135 or anti-PNf 04-7 resulted in intensified staining of the 170 kDa polypeptide by phosphorylation-dependent anti-nPNf (Figs. 3 and 4, Table 1). Although there appeared, in addition to the 170 kDa polypeptide, two minor bands at apparent molecular weights of 145 and 130 kDa, the products of extensive proteolysis seen on incubation with phosphatase of nonpurified cytoskeletal preparations were not formed.
Discussion
The data show that upon dephosphorylation, Nf-H in cytoskeletal preparations is degraded rapidly. A broad spectrum of relatively low molecular weight products forms which are partially released into solution. These products retain immunoreactivity with monoclonal anti-nPNf. Whether they also contain intact sequences corresponding to the epitopes reactive with anti-PNf, could not be determined
157 because, as shown by hapten inhibition (Sternberger and Sternberger 1983), the presence of phosphate is required in neurofilaments for reaction with anti-PNf. The protease involved in this breakdown could either be a contaminant of the phosphatase used or it could be endogenous to the cytoskeletal preparation. The former possibility is enhanced by the finding of a small peak of unknown composition on high performance liquid chromatography of E. coli phosphatase, in addition to the main phosphatase peak (unpublished). However, proteolysis did not occur either when affinity-purified Nf-H or when the chymotryptic fragment of Nf-H were incubated with phosphatase. Thus, it appears that the protease involved is endogenous to the cytoskeletal preparation and not a contaminant of the phos= phatase used. However, the protease is not intrinsic to the Nf-H structure itself. Since proteolysis only occurs upon treatment with phosphatase, it seems that at least one function of phosphorylation of Nf-H is its protection against proteolysis. It is unlikely that this is the most important function of neurofilament phosphorylation. With regard to the absence of proteolysis in affinity-purified Nf-H, one may have to consider the possibility that sodium dodecyl sulfate (SDS) treatment, incumbent to the procedure, may have made Nf-H resistant to proteolysis. This possibility is unlikely since in SDS-treated cytoskeletal preparations trypsin abolishes immunoreactivity with anti-PNf and anti-nPNf (unpublished), while in neurofilaments not treated by SDS trypsin treatment retains immunoreactivity with anti-PNf. Trypsin abolishes immunoreactivity with anti-nPNf, but only in nonphosphorylated neurofilaments and not in phosphorylated forms (Sternberger and Sternberger 1983, 1986). Thus, it seems likely that SDS treatment, if anything, makes neurofilaments more susceptible to proteolysis rather than more resistant. In any event the resistance to dephosphorylation-dependent protease exhibited by the chymotryptic fragment of Nf-H, which had not been exposed to SDS, provides independent evidence for the absence of the protease from the phosphatase preparation. The endogenous protease involved here is divalent cation-independent and differs, therefore, from the calcium-dependent neutral protease (CANP) described by Schlaepfer et al. (1984a, b) and Trojanowski et al. (1984). CANP is important in axonal breakdown resulting from calcium influx in nerve injury. Even in the absence of injury, this protease is believed to play a role in the physiologic breakdown of neurofilaments upon reaching nerve terminals in slow axonal transport (Zimmerman and Schlaepfer 1984). Since terminal axons appear to be largerly phosphorylated, it is likely that CANP acts on phosphorylated forms of neurofilaments. Proteolysis by CANP is limited to yield fragments that may well have biologic activity (Zimmerman and Schlaepfer 1984). In contrast, the protease described here is calcium-independent and acts only on nonphosphorylated forms of neurofilaments. It is unknown whether this protease has physiologic significance. The proteolysis is extensive, and it is, therefore, less likely that its products have biologic activity than in the case of CANP. What appears to be more important is the finding that phosphorylation protects neurofilamerits against degradation by enzymes, such as the protease encountered in the present communication. We have previously shown that neurofilament phosphorylation shields epitopes
158 against reaction with anti-nPNf (Sternberger and Sternberger 1983, 1986). Dephosphorylation unravels these sites. Furthermore, the epitopes reactive with anti-nPNf are protected in phosphorylated neurofilaments against the effect of trypsin, while in the nonphosphorylated form they are destroyed by trypsin. As a consequence, imrnunocytochemical staining of anti-nPNf can be abolished by trypsin and subsequently restored by phosphatase. These data suggested that phosphorylation mediated a conformational change in neurofilaments that restricted the accessibility of certain regions to antibody. The protection of neurofilaments by phosphorylation against proteolysis by calcium-independent endogenous protease as described in the present paper, provides additional suggestion towards conformational change of Nf-H mediated by phosphorylation. By electron microscopy, neurofilaments are seen in axons, neuronal cell bodies and dendrites (Peters et al. 1976). Toyoshima et al. (1984) detected no 200 kDa protein in perikarya and the middle component of the neurofilament triplet (Nf-M) had a lower isoelectric point and lower apparent molecular weight in perikarya than in axonal neurofilaments. Immunocytochemically, anti-PNf do not react in perikarya, but anti-nPNF do. However, while in electron microscopy anti-PNf localizes on intermediate filaments in axons, anti-nPNf provides a diffuse localization in perikarya (Langley et al. 1985). Apparently neurofilaments are not yet assembled as intermediate filaments when they appear in nonphosphorylated form in perikarya. It remains to be determined whether phosphorylation itself is necessary for assembly of neurofilaments from individual components of the triplet into morphologically distinguishable intermediate filaments, or whether phosphorylation occurs after assembly and mediates further changes in tertiary structure. In any event, it appears that conformational rearrangement of neurofilaments is a significant part of the function of neurofilament phosphorylation, of which protection against proteolysis is only a minor aspect.
References Bader, M.F., E. Georges, W.E. Mushynski and J.M. Trifaro, Neurofilament proteins in cultured chromaffin cells, J. Neurochem., 43 (1984) 1180-1193. Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. Carden, M.J., W,W. Schlaepfer and V.M.-Y. Lee, The structure, biochemical properties and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state, J. Biol. Chem., 260 (1985) 9805-9807. Chiu, F.-C. and W.T. Norton, Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: dye-binding characteristics and amino acid compositions, J. Neurochem., 39 (1982) 1252-1260. Chiu, F.-C., W.T. Norton and K.L. Fields, The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast type filament protein, vimentin, J. Neurochem., 37 (1981) 147-155. Eagles, P.A.M., D.S. Gilbert and A. Maggs, The location of phosphorylation sites and Ca++-dependent proteolytic cleavage sites on the major neurofilament polypeptides from Myxicola infundibulum, Biochemistry, 16 (1981) 101-111.
159 Eng, L.F. and S.J. DeArmond, Immunochemistry of glial fibrillary acidic (GFA) protein. In: H.M. Zimmerman (Ed.), Progress in Neuropathology, Vol. 5, Raven Press, New York, 1983, pp. 19-39. Fazekas de St. Groth, S. and D. Scheidegger, Production of monoclonal antibodies: strategy and tactics, J. Immunol. Methods, 35 (1980) 1-21. Geisler, N., E. Kaufmann and K. Weber, Protein chemical characterization of three structurally distinct domains along the protofilanaent unit of desmin 10 nm filaments, Cell, 30 (1982) 277-286. Geisler, N., E. Kaufmann, S. Fischer, U. Plessmann and K. Weber, Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins, EMBO J., 2 (1983) 1285-1302. Geisler, N., S. Fischer, J. Vandekerckhove, J. Van Damme, U. Plessmann and K. Weber, Protein-chemical characterization of Nf-H, the largest mammalian neurofilament component intermediate filament-type sequences followed by a unique carboxy-terminal extension, EMBO J., 4 (1985) 57-63. Goldstein, M.E., L.A. Sternberger and N.H. Sternberger, Microheterogeneity ('neurotypy') of neurofilament proteins, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 3101-3105. Goldstein, M.E., L.A. Sternberger and N.H. Sternberger, Varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (Nf-H), J. Neuroimmunol., 14 (1987) 135-148. Julien, J.-P. and W.E. Mushynski, Multiple phosphorylation sites in mammalian neurofilament polypeptides, J. Biol. Chem., 257 (1982) 10467-10470. Knapp, R.J. and L.A. Sternberger, Preparation and characterization of a monoclonal antibody to LHRH, J. Neuroimmunol., 6 (1984) 361-371. Knapp, R.J. and L.A. Sternberger, Isolation and characterization of a new peptide from hypothalamus and pituitary using a monoclonal antibody to LHRH, J. Neuroimmunol., 11 (1986) 335-351. KShler, J.G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256 (1976a) 495-497. KShler, J.G. and C. Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol., 6 (1976b) 511-519. Laemmli, U.-K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. Langley, K., N.H. Sternberger and L.A. Sternberger, Immunoelectron microscopy of neurofilament proteins with two monoclonal antibodies which distinguish different neuronal populations in the cerebellum, Biol. Cell, 53 (1985) 15a. Lowry, G.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Peters, A., S.L. Palay and H. DeF. Webster, The Fine Structure of the Nervous System: The Neuron and Supporting Cells, Sanders, Philadelphia, PA, 1976. Schlaepfer, W.W., C. Lee, J.Q. Trojanowski and V.M.-Y. Lee, Persistence of immunoreactive neurofilament protein breakdown products in transected rat sciatic nerve, J. Neurochem., 43 (1984) 857-864. Schlaepfer, W.W., C. Lee, V.M.-Y. Lee and U.-J.P. Zimmerman, An irnmunoblot study of neurofilament degradation in situ and during calcium-activated proteolysis, J. Neurochem., 44 (1985) 502-509. Sternberger, L.A., Immunocytochemistry, 3rd edn., John Wiley, New York, 1986 (324 pp.). Sternberger, L.A. and N.H. Sternberger, Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 6126-6130. Sternberger, L.A. and N.H. Sternberger, Enzymatic alterations of neurofilament staining patterns, J. Neuropathol. Exp. Neurol., 45 (1986) 346. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis and H.G. Meyer, The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. Sternberger, L.A., L.W. Harwell and N.H. Sternberger, Neurotypy: regional individually in rat brain detected by immunocytochemistry with monoclonal antibodies, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 1326-1330. Toyoshima, I., M. Satake and T. Miyatake, Differences in the neurofilament proteins between the perikaryon and axon of the bovine spinal ganglion, Biomed. Res., 5 (1984) 459-464.
160 Towbin, H., T. Staeblin and J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A., 76(9) (1979) 4350-4354. Trojanowski, J.Q., V.M.-Y. Lee and W.W. Schlaepfer, Neurofilament breakdown products in degenerating rat and human peripheral nerves, Ann. Neurol., 16 (1984) 349-355. Weber, K., G. Shaw, M. Osborn, E. Debus and N. Geisler, Neurofilaments, a subclass of intermediate filaments: structure and expression, Cold Spring Harbor Symp. Quant. Biol., 47 (1983) 717-729. Zimmerman, U.-J.P. and W.W. Schlaepfer, Calcium-activated neutral protease (CANP) in brain and other tissues, Prog, Neurobiol., 23 (1984) 63-78.
149
Elsevier JNI 00458
Phosphorylation protects neurofilaments against proteolysis Margi E. Goldstein *, Nancy H. Sternberger and Ludwig A. Sternberger Center for Brain Research, University of Rochester School of Medicine and Dentistry, Rochester, N Y 14642, and Departments of A natomy, Neurology and Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. (Received 1 April 1986) (Revised, received 25 August 1986) (Accepted 26 August 1986)
Key words: Neurofilament conformation; Monoclonal antibodies; Peroxidase-monoclonal antiperoxidase technique; Neurofilament protease, calcium independent
Summary During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that (1) phosphorylated neurofilaments are protected against proteolysis, and (2) dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.
* In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience. Supported by National Science Foundation grant BNS-8506474 and by National Institutes of Health grants NS 21681 (Javits Award), NS 17652, and NIMH F31 MH09100. Address for correspondence: Dr. Nancy Sternberger, Department of Anatomy, University of Maryland School of Medicine, 655 W. Baltimore Street, Ba/dmore, MD 21201, U.S.A. 0165-5728/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
150 Introduction
The finding that some monoclonal antibodies to neurofilaments react with phosphorylated epitopes has been dependent upon (a) inhibition of the antibodies by inorganic phosphate, acting as a hapten (Sternberger and Sternberger 1983), (b) failure of these antibodies to react with the nonphosphorylated 170 kDa component of the heavy neurofilament peptide (Nf-H) (Goldstein et al. 1987), and (c) failure to react with phosphatase-treated neurofilaments (Sternberger and Sternberger 1983). That the phosphatase effects, indeed, were due to phosphatase rather than any other component in the enzyme preparation was surmised from their inhibition by inorganic phosphate, an inhibitor of phosphatase. However, more recently we observed that incubation of neurofilaments with phosphatase produced a number of lower molecular weight forms in addition to those that can be attributed to dephosphorylation alone. The present paper examines these effects.
Materials and methods
Monoclonal antibodies Monoclonal antibodies were produced by the method of Ki3hler and Milstein (1976a, b) and Fazekas de St. Groth and Scheidegger (1980) as described previously (Sternberger et al. 1982). Antibodies 02-135 and 06-32 reacted with nonphosphorylated neurofilaments (anti-nPNf), antibodies 03-44, 06-17, 04-7 and 07-5 with phosphorylated neurofilaments (anti-PNf), and antibody 02-40 with a nonphosphorylated epitope in both, nonphosphorylated and phosphorylated neurofilaments.
Cytoskeletal preparation Cytoskeletal proteins were prepared by the method of Chiu et al. (1981, 1982) and Eng and DeArmond (1983) as described previously (Go!dstein et al. 1983). Protein concentrations were determined by the method of Lowry et al. (1951) or Bradford (1976).
Gel electrophoresis One-dimensional gels were set up by the method of Laemmli (1970) as described previously (Goldstein et al. 1983). The separating gels contained 7.5% acrylamide. The gels were either stained in 0.125% Coomassie blue R-250 in 50% methanol/10% acetic acid, or immunoblotted by the method of Towbin et al. (1979) as described previously (Goldstein et al. 1983) using 0.025 M Tris-HC1, pH 8.03/0.05 M NaC1/0.19 M glycine/10% methanol.
Immunocytochemistry Immunoblots were stained immunocytochemically (Sternberger et al. 1970) using mouse peroxidase-antiperoxidase complex made from monoclonal antiperoxidase (Sternberger 1986).
OF NEUROFILAMENTS.
SUMMARY OF IMMUNOBLOTS
P
200
170 150 130 LMF
Anti-PNf
(03-44, 06-17, 04-7, 07-5) -
+
Masked Masked + + + +
++++ ++++
Masked Masked ++++
+++
++++
+
+++
+
_.
_
+
_
_
+
+ +
+ +
+
+
+
++
+
++++ +++
++++
+
+
++
++++
+
++
++++
P+Ap
P
++
++++
+ +
_
+ +
++++
++
-
++
+
-
-
-
+
+
+
+
Masked Masked
++
--
Masked Masked ++++
+
.
Soluble fraction
B
.
+4
+++
+ +
-
+ +
++++
+ +
.
+
P+Pi
.
.
+
++
+
_
+ +
++
+
-
+
.
.
P+Ap
+ + + + + + .
-
B
fragment
+
+
B
+ + +
--
+
+ +
. . .
--
.
--
+
+ + + +
. .
++++
-
P
. .
+ +
--
+
4 +
+
+
P+Pi
Affinity-purified Nf-H
+ . q: + + + . . .
-
P
Chymotryptic
+ +
_
+
+ +
+
+
P+Ap
B, buffer; P, p h o s p h a t a s e ; P + Pi, p h o s p h a t a s e in p h o s p h a t a s e buffer; P + Ap, p h o s p h a t a s e plus a m i n o p h y l l i n e ; n P N f , n o n p h o s p h o r y l a t e d n e u r o f i l a m e n t s ; PNf, phosphorylated neurofilaments. a A n t i b o d i e s to n P N f epitopes m a s k e d b y p h o s p h o r y l a t i o n . b A n t i b o d y to n P N f epitopes accessible in n P N f as well as PNf. c Lower molecular weight fragments.
-
++++
+ + -
150 130 LMF
-
150 130 LMF c
++++ ++
+++
170
200 170
++++
200
Insoluble residue
B
P+Pi
T r e a t m e n t of p r o t e i n s
fraction Cytoskeletal p r o t e i n s (kDa)
Phosphorylationindependent anti-nPNf (02-40) b
(02-135, 06-32) "
Phosphorylationdependent anti-nPNf
antibodies
Monoclonal MW
E s t i m a t e s of staining are a v e r a g e s w h e r e different a n t i b o d i e s o f the s a m e g r o u p e x h i b i t e d d i f f e r e n t d e g r e e s of staining ( m i c r o h e t e r o g e n e i t y , G o l d s t e i n et al. 1983, 1987). W e a k e r staining n o n t r i p l e t n e u r o f i l a m e n t b a n d s with a p p a r e n t m o l e c u l a r w e i g h t s of 178, 164, 145 a n d 124 k D a not included.
PROTEOLYSIS AFTER DEPHOSPHORYLATION
TABLE 1
~ O n -
-- N
N
N
2
¸-¸
II
C '~
N
M
U m
Fig. 1. Immunoblots of neurofilaments degraded after dephosphorylation. Cytoskeletal preparations were incubated at 32°C for 20 h, and the proteins separated into precipitates (a and b) and supernatants (c and d). Incubation in Tris buffer (a and c, lanes 1), Tris buffer plus 250 ~ g / m l alkaline phosphatase (a and c, lanes 2), sodium phosphate buffer plus 250/~g/ml alkaline phosphatase (b and d, lanes 1), or Tris buffer plus 250 ~ g / m l alkaline phosphatase and aminophylline (b and d, lanes 2). Immunocytochemical staining with the antibodies listed. Molecular weight standards are myosin, 200 kDa; fl-galactosidase, 116.5 kDa; phosphorylase b, 92.5 kDa, bovine serum albumin, 66 kDa and ovalbumin, 45 kDa.
153
lmmunoprecipitation Neurofilaments were precipitated with anti-nPNf 02-135 and anti-PNf 04-7 by the method of Bader et al. (1984) as described in the accompanying paper (Goldstein et al. 1987). Anti-nPNf and anti-PNf were replaced with monoclonal antibody to L H R H ( K n a p p and Sternberger 1984, 1986) in control precipitations.
Chymotrypsin digestion Cytoskeletal proteins, at concentrations of 3 m g / m l in 0.1 M Tris-HC1, p H 8.0, were incubated with 1 # g / m l chymotrypsin at 32°C for 1 h. Phenylmethylsulfonyl fluoride (PMSF) was added to stop the reaction and the proteins were centrifuged at 15 600 × g in an Eppendorf microcentrifuge for 30 rain. Precipitates and supernatants were electrophoresed as such or treated with E. coli alkaline phosphatase prior to electrophoresis as described below.
Enzymatic dephosphorylation Cytoskeletal proteins, immunoprecipitated Nf-H, or chymotryptic fragments of neurofilaments were dephosphorylated with E. coli alkaline phosphatase (Worthington) as described in the accompanying paper (Goldstein et al. 1987). Phosphatase activity was inhibited by the addition of 0.2 M sodium phosphate or 2 m M aminophylline.
Results When cytoskeletal preparations were incubated in E. coli phosphatase and the ensuring precipitates immunoblotted, staining with anti-nPNf revealed N f - H as a
Fig. 2. Stability of dephosphorylated chymotryptic fragment. Cytoskeletal proteins treated with chymotrypsin and separated into precipitate (lane 1) and supernatant (lanes 2 and 3) were incubated in Tris buffer (lanes 1 and 2) or 250 #g/ml alkaline phosphatase at 32°C for 20 h (lane 3), transblotted and stained immunocytochemicallywith anti-nPNf 02-135. The molecular weight of the 160 kDa polypeptide released into the supernatant (*) became reduced to 130 kDa (**), upon treatment with phosphatase.
154 0
t M,+
~
w
g
+
b
=
+++
+P <
+++
2 1 2 ..... 2 +++++++++ ++ +++~++++++++++++++~++++ +
.... t
+
+
+ ;
++++++++++ +
+
....+ ++.... +
++++++++ + ++++++++
++ +..... +t +++++ +
.............. + ++++++++ ++.... ++++++ ++++++ ++ ++++++ .....
+ ++++
++
< +++++
+ +....
+ I +++
+++++ +++ ++ ++
+
+++ +++++ ++++++++++++++++ +
++++++++++?+++ +++++++++++?+++++ ++++++++++;+
Fig. 3. Stability of neurofilaments upon dephosphorylation after affinity purification with anti-nPNf. Cytoskeletal proteins immunoprecipitated by anti-nPNf 02-135 were incubated at 32°C for 20 h in Tris buffer (a), Tris buffer plus 250 ~ g / m l alkaline phospliatase (b), sodium phosphatase buffer plus 250 / t g / m l alkaline phosphatase (c, lanes 1), or Tris buffer plus 250 ~tg/ml alkaline phosphatase and aminophylline (c, lanes 2). The transblots were stained immunocytochemically with anti-nPNf 02-135, 06-32 and 02-40, and anti-PNf 03-44, 06-17, 04-7 and 07-5. Molecular weight standards as in Fig. 1. Dephosphorylation intensifies the 170 kDa band (**) and reveals two proteins at 145 (*) and 130 kDa (*).
155 O
-t
N
N
k
b
)
~
iiii~iiiii~ iii~i~i
'7
~i!!~!i!i!i~i ~ii
i~i~:~iii
iliii
!~
:II~'!~!!~!i ~ ii~I,!ili!~!iiiii!iiii!!~!!i!iiiii~!i! !! ~! ~i~i!!iiiii !!i !!!!~!iiiii~!!!i!!i!ii!!!!~!iiiii~i iii!~iiiiiii!!~!i!ili~i!~!!i!iiiiiii!ii!!!!ii!ii!~ ~ ~i~ ~ ~~!i~,~~ili!~i"~!i~ Fig. 4. Stability of neurofilaments after affinity purification with anti-PNf. Cytoskeletal proteins immunoprecipitated by anti-PNf 04-7 were incubated at 32°C for 20 h in Tris buffer (a), Tris buffer plus 250 # g / m l alkaline phosphatase (b), sodium phosphate buffer plus 2 5 0 / t g / m l alkaline phosphatase (c, lanes 1), or Tris buffer plus 250 / t g / m l alkaline phosphatase and aminophylline (d, lanes 2). The transblots were stained immunocytochemically with anti-nPNf 02-135, 06-32 and 02-40, and anti-PNf 03-44, 06-17, 04-7 and 07-5. Molecular weight standards as in Fig. 1. Dephosphorylation intensifies the 170 kDa band (**) and reveals two proteins at 145 (*) and 130 kDa (*).
156 broad band that included lower molecular weight forms and extended over most of the immunoblot strip (Fig. la, Table 1). Overall staining was also intensified compared to controls incubated in the absence of phosphatase. Staining with anti-PNf, on the other hand, was reduced, as expected, after incubation with phosphatase (Sternberger and Sternberger 1983), and the breakdown products could not be detected. Cytoskeletal preparations incubated in buffer did not reveal lower molecular weight breakdown products nor did breakdown occur when inhibitors of phosphatase, such as inorganic phosphate or aminophylline, were added to the phosphatase (Fig. lb). Proteolysis upon phosphatase treatment was not affected by divalent metal-chelating agents (EDTA, EGTA) nor did inhibitors of proteases, such as leupeptin, PMSF or peptastatin affect the breakdown. Cytoskeletal preparations incubated in buffer released into solution no significant amount of protein detectable by immunoblotting with anti-nPNf 02-135 and 06-32 (Fig. lc). On the other hand, incubation in phosphatase solubilized a considerable portion of the Nf-H breakdown products, as well as traces of the 170 kDa component corresponding to the dephosphorylated fraction of intact Nf-H (Julien and Mushynski 1982; Carden et al. 1985). Again, release of material into solution by phosphatase treatment was inhibited by inorganic phosphate and aminophylline (Fig. ld). o n immunoblots of chymotrypsin-digested neurofilaments, all seven antibodies stained the 160 kDa fragment released into the supernatant. None of the antibodies stained the 40 kDa chymotryptic fragment associated with the insoluble neurofilament core (Geisler et al. 1982, 1983; Weber et al. 1983). In addition to the 160 kDa chymotryptic product which is stained by all seven antibodies, anti-nPNf stained a protein band at an apparent molecular weight of 130 kDa. When the 160 kDa fragment was incubated with alkaline phosphatase, its apparent molecular weight became reduced, but no additional degradative products were stained (Fig. 2, Table 1). Incubation with E. coli alkaline phosphatase of Nf-H immunoprecipitated by anti-nPNf 02-135 or anti-PNf 04-7 resulted in intensified staining of the 170 kDa polypeptide by phosphorylation-dependent anti-nPNf (Figs. 3 and 4, Table 1). Although there appeared, in addition to the 170 kDa polypeptide, two minor bands at apparent molecular weights of 145 and 130 kDa, the products of extensive proteolysis seen on incubation with phosphatase of nonpurified cytoskeletal preparations were not formed.
Discussion
The data show that upon dephosphorylation, Nf-H in cytoskeletal preparations is degraded rapidly. A broad spectrum of relatively low molecular weight products forms which are partially released into solution. These products retain immunoreactivity with monoclonal anti-nPNf. Whether they also contain intact sequences corresponding to the epitopes reactive with anti-PNf, could not be determined
157 because, as shown by hapten inhibition (Sternberger and Sternberger 1983), the presence of phosphate is required in neurofilaments for reaction with anti-PNf. The protease involved in this breakdown could either be a contaminant of the phosphatase used or it could be endogenous to the cytoskeletal preparation. The former possibility is enhanced by the finding of a small peak of unknown composition on high performance liquid chromatography of E. coli phosphatase, in addition to the main phosphatase peak (unpublished). However, proteolysis did not occur either when affinity-purified Nf-H or when the chymotryptic fragment of Nf-H were incubated with phosphatase. Thus, it appears that the protease involved is endogenous to the cytoskeletal preparation and not a contaminant of the phos= phatase used. However, the protease is not intrinsic to the Nf-H structure itself. Since proteolysis only occurs upon treatment with phosphatase, it seems that at least one function of phosphorylation of Nf-H is its protection against proteolysis. It is unlikely that this is the most important function of neurofilament phosphorylation. With regard to the absence of proteolysis in affinity-purified Nf-H, one may have to consider the possibility that sodium dodecyl sulfate (SDS) treatment, incumbent to the procedure, may have made Nf-H resistant to proteolysis. This possibility is unlikely since in SDS-treated cytoskeletal preparations trypsin abolishes immunoreactivity with anti-PNf and anti-nPNf (unpublished), while in neurofilaments not treated by SDS trypsin treatment retains immunoreactivity with anti-PNf. Trypsin abolishes immunoreactivity with anti-nPNf, but only in nonphosphorylated neurofilaments and not in phosphorylated forms (Sternberger and Sternberger 1983, 1986). Thus, it seems likely that SDS treatment, if anything, makes neurofilaments more susceptible to proteolysis rather than more resistant. In any event the resistance to dephosphorylation-dependent protease exhibited by the chymotryptic fragment of Nf-H, which had not been exposed to SDS, provides independent evidence for the absence of the protease from the phosphatase preparation. The endogenous protease involved here is divalent cation-independent and differs, therefore, from the calcium-dependent neutral protease (CANP) described by Schlaepfer et al. (1984a, b) and Trojanowski et al. (1984). CANP is important in axonal breakdown resulting from calcium influx in nerve injury. Even in the absence of injury, this protease is believed to play a role in the physiologic breakdown of neurofilaments upon reaching nerve terminals in slow axonal transport (Zimmerman and Schlaepfer 1984). Since terminal axons appear to be largerly phosphorylated, it is likely that CANP acts on phosphorylated forms of neurofilaments. Proteolysis by CANP is limited to yield fragments that may well have biologic activity (Zimmerman and Schlaepfer 1984). In contrast, the protease described here is calcium-independent and acts only on nonphosphorylated forms of neurofilaments. It is unknown whether this protease has physiologic significance. The proteolysis is extensive, and it is, therefore, less likely that its products have biologic activity than in the case of CANP. What appears to be more important is the finding that phosphorylation protects neurofilamerits against degradation by enzymes, such as the protease encountered in the present communication. We have previously shown that neurofilament phosphorylation shields epitopes
158 against reaction with anti-nPNf (Sternberger and Sternberger 1983, 1986). Dephosphorylation unravels these sites. Furthermore, the epitopes reactive with anti-nPNf are protected in phosphorylated neurofilaments against the effect of trypsin, while in the nonphosphorylated form they are destroyed by trypsin. As a consequence, imrnunocytochemical staining of anti-nPNf can be abolished by trypsin and subsequently restored by phosphatase. These data suggested that phosphorylation mediated a conformational change in neurofilaments that restricted the accessibility of certain regions to antibody. The protection of neurofilaments by phosphorylation against proteolysis by calcium-independent endogenous protease as described in the present paper, provides additional suggestion towards conformational change of Nf-H mediated by phosphorylation. By electron microscopy, neurofilaments are seen in axons, neuronal cell bodies and dendrites (Peters et al. 1976). Toyoshima et al. (1984) detected no 200 kDa protein in perikarya and the middle component of the neurofilament triplet (Nf-M) had a lower isoelectric point and lower apparent molecular weight in perikarya than in axonal neurofilaments. Immunocytochemically, anti-PNf do not react in perikarya, but anti-nPNF do. However, while in electron microscopy anti-PNf localizes on intermediate filaments in axons, anti-nPNf provides a diffuse localization in perikarya (Langley et al. 1985). Apparently neurofilaments are not yet assembled as intermediate filaments when they appear in nonphosphorylated form in perikarya. It remains to be determined whether phosphorylation itself is necessary for assembly of neurofilaments from individual components of the triplet into morphologically distinguishable intermediate filaments, or whether phosphorylation occurs after assembly and mediates further changes in tertiary structure. In any event, it appears that conformational rearrangement of neurofilaments is a significant part of the function of neurofilament phosphorylation, of which protection against proteolysis is only a minor aspect.
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159 Eng, L.F. and S.J. DeArmond, Immunochemistry of glial fibrillary acidic (GFA) protein. In: H.M. Zimmerman (Ed.), Progress in Neuropathology, Vol. 5, Raven Press, New York, 1983, pp. 19-39. Fazekas de St. Groth, S. and D. Scheidegger, Production of monoclonal antibodies: strategy and tactics, J. Immunol. Methods, 35 (1980) 1-21. Geisler, N., E. Kaufmann and K. Weber, Protein chemical characterization of three structurally distinct domains along the protofilanaent unit of desmin 10 nm filaments, Cell, 30 (1982) 277-286. Geisler, N., E. Kaufmann, S. Fischer, U. Plessmann and K. Weber, Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins, EMBO J., 2 (1983) 1285-1302. Geisler, N., S. Fischer, J. Vandekerckhove, J. Van Damme, U. Plessmann and K. Weber, Protein-chemical characterization of Nf-H, the largest mammalian neurofilament component intermediate filament-type sequences followed by a unique carboxy-terminal extension, EMBO J., 4 (1985) 57-63. Goldstein, M.E., L.A. Sternberger and N.H. Sternberger, Microheterogeneity ('neurotypy') of neurofilament proteins, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 3101-3105. Goldstein, M.E., L.A. Sternberger and N.H. Sternberger, Varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (Nf-H), J. Neuroimmunol., 14 (1987) 135-148. Julien, J.-P. and W.E. Mushynski, Multiple phosphorylation sites in mammalian neurofilament polypeptides, J. Biol. Chem., 257 (1982) 10467-10470. Knapp, R.J. and L.A. Sternberger, Preparation and characterization of a monoclonal antibody to LHRH, J. Neuroimmunol., 6 (1984) 361-371. Knapp, R.J. and L.A. Sternberger, Isolation and characterization of a new peptide from hypothalamus and pituitary using a monoclonal antibody to LHRH, J. Neuroimmunol., 11 (1986) 335-351. KShler, J.G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256 (1976a) 495-497. KShler, J.G. and C. Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol., 6 (1976b) 511-519. Laemmli, U.-K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. Langley, K., N.H. Sternberger and L.A. Sternberger, Immunoelectron microscopy of neurofilament proteins with two monoclonal antibodies which distinguish different neuronal populations in the cerebellum, Biol. Cell, 53 (1985) 15a. Lowry, G.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Peters, A., S.L. Palay and H. DeF. Webster, The Fine Structure of the Nervous System: The Neuron and Supporting Cells, Sanders, Philadelphia, PA, 1976. Schlaepfer, W.W., C. Lee, J.Q. Trojanowski and V.M.-Y. Lee, Persistence of immunoreactive neurofilament protein breakdown products in transected rat sciatic nerve, J. Neurochem., 43 (1984) 857-864. Schlaepfer, W.W., C. Lee, V.M.-Y. Lee and U.-J.P. Zimmerman, An irnmunoblot study of neurofilament degradation in situ and during calcium-activated proteolysis, J. Neurochem., 44 (1985) 502-509. Sternberger, L.A., Immunocytochemistry, 3rd edn., John Wiley, New York, 1986 (324 pp.). Sternberger, L.A. and N.H. Sternberger, Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 6126-6130. Sternberger, L.A. and N.H. Sternberger, Enzymatic alterations of neurofilament staining patterns, J. Neuropathol. Exp. Neurol., 45 (1986) 346. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis and H.G. Meyer, The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. Sternberger, L.A., L.W. Harwell and N.H. Sternberger, Neurotypy: regional individually in rat brain detected by immunocytochemistry with monoclonal antibodies, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 1326-1330. Toyoshima, I., M. Satake and T. Miyatake, Differences in the neurofilament proteins between the perikaryon and axon of the bovine spinal ganglion, Biomed. Res., 5 (1984) 459-464.
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