The properties regulating the supramolecular organization of neural intermediate filament (NIF) networks have been investigated in cultured dorsal root ganglion (DRG) neurons. The studies described take advantage of the ability of endogenous NIF to incorporate purified biotinylated neurofilament triplet (NFT) proteins, NF-L, NF-M and NF-H. When injected at concentrations of 0.8-1.0 mg/ml injection buffer, each of these proteins is incorporated without perturbing the endogenous NIF network. However, at progressively higher concentrations, NF-H induces the aggregation and accumulation of NIF in the cell body. Subsequent to the induction of these aggregates, numerous alterations in the cytoarchitecture of neurons can be detected. The latter occur in a temporal sequence which appears to begin with the fragmentation of the Golgi complex. At later times, accumulation of mitochondria within the proximal region of neurites, peripheralization of the nucleus, and a significant decrease in neurite caliber become obvious. After longer time periods, the NIF aggregates are seen to react with an antibody which reveals abnormally phosphorylated NF-H. These observations demonstrate that an imbalance in the normal stoichiometric relationships among the NFT proteins rapidly alters the supramolecular organization of the NIF network. These changes most likely reflect the normal functions of neurofilaments in cell shape and the organization and cytoplasmic distribution of membranous organelles. Interestingly, virtually all of these changes closely resemble those which have been reported in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). These findings suggest that cultured neurons can be used as models for more precisely defining the relationships between the formation of NIF aggregates and the sequence of cytopathological events which typify neurodegenerative diseases.
REFERENCES
Aebi
U.
, Haner
M.
, Troncoso
J.
, Eichner
R.
, Engel
A.
(1988
). Unifying principles in intermediate filament (IF) structure and assembly.
Protoplasma
145
, 73
–81
Angelides
K. J.
, Smith
K. E.
, Takeda
M.
(1989
). Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.
J. Cell Biol
108
, 1495
–1506
Arakawa
O.-M.
, Narahashi
T.
(1992
). Chloride current induced by alcohols in rat dorsal root ganglion neurons.
Brain Res
578
, 275
–281
Balin
B. J.
, Lee
V. M.-Y.
(1991
). Individual neurofilament subunits reassembled in vitro exhibit unique biochemical, morphological and immunological properties.
Brain Res
556
, 196
–208
Bennett
G. S.
, Tapscott
S. J.
, Kleinbart
F. A.
, Antin
P. B.
, Holtzer
H.
(1981
). Different proteins associated with 10-nanometer filaments in cultured chick neurons and non-neuronal cells.
Science
212
, 567
–569
Bennett
G. S.
, Tapscott
S. J.
, DiLullo
C.
, Holtzer
H.
(1984
). Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons.
Brain Res
304
, 291
–302
Bizzi
A.
, Crane
R. C.
, Autilio-Gambetti
L.
, Gambetti
P.
(1984
). Aluminum effect on slow axonal transport: a novel impairment of neurofilament transport.
J. Neurosci
4
, 722
–731
Bowling
A. C.
, Schulz
J. S.
, Brown
R. H.
, Beal
M. F.
(1993
). Superoxide dismutase activity, oxidative damage, and mitochondrial energy metabolism in familial and sporatic amyotrophic lateral sclerosis.
J. Neurochem
61
, 2322
–2325
Bradford
M. M.
(1976
). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem
72
, 248
–254
Breuer
A. C.
, Lynn
M. P.
, Atkinson
M. B.
, Chou
S. M.
, Wilbourn
A. J.
, Marks
K. E.
, Culver
J. E.
, Fleegler
E. J.
(1987
). Fast axonal transport in amyotrophic lateral sclerosis: an intra-axonal organelle traffic analysis.
Neurology
37
, 738
–748
Carden
M. J.
, Schlaepfer
W. W.
, Lee
V. M.-Y.
(1985
). The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state.
J. Biol. Chem
260
, 9805
–9817
Carpenter
S.
(1968
). Proximal axonal enlargement in motor neuron disease.
Neurology
18
, 841
–851
Ching
G. Y.
, Liem
R. K. H.
(1993
). Assembly of Type IV neuronal intermediate filaments in non-neuronal cells in the absence of pre-existing cytoplasmic intermediate filaments.
J. Cell Biol
122
, 1323
–1335
Chiu
F.-C.
, Korey
B.
, Norton
W. T.
(1980
). Intermediate filaments frombovine, rat and human CNS: mapping analysis of major proteins.
J. Neurochem
34
, 1149
–1159
Collard
J.-F.
, Côte
F.
, Julien
J.-P.
(1995
). Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis.
Nature
375
, 61
–64
Cork
L. C.
, Sternberger
N. H.
, Sternberger
L. A.
, Casanova
M. F.
, Strubl
R. G.
, Price
D. L.
(1986
). Phosphorylated neurofilament antigens in neurofibrillary tangles in Alzheimer's disease.
J. Neuropath. Exp. Neurol
45
, 56
–64
Côte
F.
, Collard
J.-F.
, Julien
J.-P.
(1993
). Progressive neuronopathy in transgenic mice expressing the neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis.
Cell
73
, 35
–46
deWaegh
S. M.
, Lee
V. M.-Y.
, Brady
S. T.
(1992
). Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells.
Cell
68
, 451
–463
Friede
R. L.
, Samorajski
T.
(1970
). Axonal caliber related to neurofilaments and microtubules in sciatic nerve fibres of rats and mice.
Anat. Rec
167
, 379
–388
Gardner
E. E.
, Dahl
D.
, Bignami
A.
(1984
). Formation of 10nm filaments from the 150K-Dalton neurofilament protein in vitro.
J. Neurosci. Res
11
, 145
–155
Gasser
H. S.
, Grundfest
H.
(1939
). Axon diameters in relation to spike dimensions and the conduction velocity in mammalian A fibers.
Am. J. Physiol
127
, 393
–414
Geisler
N.
, Weber
K.
(1981
). Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments.
J. Mol. Biol
151
, 565
–571
Geisler
N.
, Kaufmann
E.
, Fischer
S.
, Plessmann
U.
, Weber
K.
(1983
). Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.
EMBO J
2
, 1295
–1302
Gonatas
N. K.
, Stieber
A.
, Mourelatos
Z.
, Chen
Y.
, Gonatas
J. O.
, Appel
S. H.
, Hays
A. P.
, Hickey
W. F.
, Hauw
J. J.
(1992
). Fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis.
Am. J. Pathol
140
, 731
–737
Griffin
J. W.
, Hoffman
P. N.
, Clark
A. W.
, Carroll
P. T.
, Price
D. L.
(1978
). Slow axonal transport of neurofilament proteins: impairment of beta, beta-iminodipropionitrile adminstration.
Science
202
, 633
–635
Hamburger
V.
, Hamilton
H. L.
(1951
). A series of normal stages in the development of the chick embryo.
J. Morphol
88
, 49
–92
Henneman
E.
, Somjen
G.
, Carpenter
D. O.
(1965
). Excitability and inhibitability of motoneurons of different sizes.
J. Neurophysiol
28
, 599
–619
Hirano
A.
, Donnenfeld
H.
, Sasaki
S.
, Nakano
I.
(1984
). Fine structured observations of neurofilamentous changes in amyotrophic lateral sclerosis.
J. Neuropathol. Exp. Neurol
43
, 461
–470
Hirokawa
N.
(1982
). The crosslinker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by quick-freeze, freeze-fracture, deep-etching method.
J. Cell Biol
94
, 129
–142
Hirokawa
N.
, Glicksman
M. A.
, Willard
M.
(1984
). Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton.
J. Cell Biol
98
, 1523
–1536
Hisanaga
S.
, Hirokawa
N.
(1988
). Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing.
J. Mol. Biol
202
, 297
–305
Hisanaga
S.
, Hirokawa
N.
(1990
). Dephosphorylation-induced interactions of neurofilaments with microtubules.
J. Biochem
265
, 21852
–21858
Hoffman
P. N.
, Lasek
R. J.
(1975
). The slow component of axonal transport: Identification of the major structural polypeptides of the axon and their generality among mammalian neurons.
J. Cell Biol
66
, 351
–366
Hoffman
P. N.
, Griffin
J. W.
, Price
D. L.
(1984
). Control of axonal caliber by neurofilament transport.
J. Cell Biol
99
, 705
–714
Hoffman
P. N.
, Thompson
G. W.
, Griffin
J.
, Price
D. L.
(1985
). Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers.
J. Cell Biol
101
, 1332
–1340
Itoh
T.
, Sobue
G.
, Ken
E.
, Mitsuma
T.
, Takahashi
A.
, Trojanowski.
J. Q.
(1992
). Phosphorylated high molecular weight neurofilament protein in the peripheral motor, sensory and sympathetic neuronal perikarya: system-dependent normal variations and changes in amyotrophic lateral sclerosis and multiple system atrophy.
Acta Neuropathol
83
, 240
–245
Kreis
T. E.
(1990
). Role of microtubules in the organization of the Golgi apparatus.
Cell Motil. Cytoskel
15
, 67
–70
Laemmli
U. K.
(1970
). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.
Nature
227
, 680
–685
Lee
G. M.
(1989
). Measurement of volume injected into individual cells by quantitative fluorescence microscopy.
J. Cell Sci
94
, 443
–447
Lee
M. K.
, Xu
Z.
, Wong
P. C.
, Cleveland
D. W.
(1993
). Neurofilaments are obligate heteropolymers in vivo.
J. Cell Biol
122
, 1337
–1350
Lee
M. K.
, Marszalek
J. R.
, Cleveland
D. W.
(1994
). A mutant neurofilament subunit causes massive, selective motor neuron death: implications for pathogenesis of human motor neuron disease.
Neuron
13
, 975
–988
Lee
V. M.-Y.
, Carden
M. J.
, Schlaepfer
W. W.
, Trojanowski
J. Q.
(1987
). Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats.
J. Neurosci
7
, 3474
–3488
Letourneau
P. C.
, Wire
J. P.
(1995
). Three dimensional organization of stable microtubules and the Golgi apparatus in the somata of developing chick sensory neurons.
J. Neurocytol
24
, 207
–223
Liem
R. K. H.
, Yen
S.-H.
, Solomon
G. D.
, Shelanski
M. L.
(1978
). Intermediate filaments in nervous tissue.
J. Cell Biol
79
, 637
–645
Liem
R. K. H.
, Hutchinson
C.
(1982
). Purification of individual components of the neurofilament triplet: filament assembly from the 70,000 dalton subunit.
Biochemistry
21
, 3221
–3226
Manetto
V.
, Sternberger
N. H.
, Perry
G.
, Sternberger
L. A.
, Gambetti
P.
(1988
). Phosphorylation of neurofilaments is altered in amyotrophic lateral sclerosis.
J. Neuropath. Exp. Neurol
47
, 642
–653
Miller
R. K.
, Khuon
S.
, Goldman
R. D.
(1993
). Dynamics of keratin assembly: exogenous Type I keratin rapidly associates with Type II keratinin vivo.
J. Cell Biol
122
, 123
–135
Minaschek
G.
, Beriter-Hahn
J.
, Bertholdt
G.
(1989
). Quantitation of the volume of liquid injected into cells by means of pressure.
Exp. Cell Res
183
, 434
–432
Miyasaka
H.
, Okabe
S.
, Ishiguro
K.
, Uchida
T.
, Hirokawa
N.
(1993
). Interaction of the tail domain of high molecular weight subunits of neurofilaments with the COOH-terminal region of tubulin and its regulation by tau protein kinase II.
J. Biol. Chem
268
, 22695
–22702
Monteiro
M. J.
, Hoffman
P. N.
, Gearhart
J. D.
, Cleveland
D. W.
(1990
). Expression of NF-L in both neuronal and non-neuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber.
J. Cell Biol
111
, 1543
–1557
Moon
H. M.
, Wisniewski
T.
, Merz
P.
, DeMartin
J.
, Wisniewski
H. M.
(1981
). Partial purification of subunits from bovine brains and studies on neurofilament assembly.
J. Cell Biol
89
, 560
–567
Mori
H.
, Kurokawa
M.
(1980
). Morphological and biochemical characterization of neurofilaments isolated from rat peripheral nerve.
Biochem. Res
1
, 24
–30
Mose-Larsen
P.
, Bravo
R.
, Fey
S. Y.
, Small
J. V.
, Celis
J. E.
(1983
). Putative association of mitochondria with a subpopulation of intermediate-sized filaments in cultured human skin fibroblasts.
Cell
31
, 681
–692
Mourelatos
Z.
, Adler
H.
, Hirano
A.
, Donnenfeld
H. D.
, Gonatas
J. O.
, Gonatas
N. K.
(1990
). Fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis revealed by organelle-specific antibodies.
Proc. Nat. Acad. Sci. USA
87
, 4393
–4395
Mourelatos
Z.
, Yachnis
A.
, Rorke
L.
, Mikol
J.
, Gonatas
N. K.
(1993
). The Golgi apparatus of motor neurons in amyotrophic lateral sclerosis.
Ann. Neurol
33
, 608
–615
Mourelatos
A.
, Hirano
A.
, Rosenquist
A. C.
, Gonatas
N. K.
(1994
). Fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis (ALS).
Am. J. Pathol
144
, 1288
–1300
Nakagawa
T.
, Chen
J.
, Zhang
Z.
, Kanai
Y.
, Hirokawa
N.
(1995
). Two distinct functions of the carboxyl-tail domain of NF-M upon neurofilament assembly: cross bridge formation and longitudinal elongation of filaments.
J. Cell Biol
129
, 411
–429
Nixon
R. A.
, Sihag
R. K.
(1991
). Neurofilament phosphorylation: a new look at regulation and function.
Trends Neurosci
14
, 501
–506
Nixon
R. A.
, Shea
T. B.
(1992
). Dynamics of neuronal intermediate filaments: a developmental perspective.
Cell Motil. Cytoskel
22
, 81
–91
Nixon
R. A.
(1993
). The regulation of protein dynamics by phosphorylation: clues to neurofibrillary pathobiology.
Brain Pathol
3
, 29
–38
Ohara
O.
, Gahra
Y.
, Miyake
T.
, Teraoka
H.
, Kitamura
T.
(1993
). Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene.
J. Cell Biol
121
, 387
–395
Okabe
S.
, Miyasaka
H.
, Hirokawa
N.
(1993
). Dynamics of the neuronal intermediate filaments.
J. Cell Biol
121
, 375
–386
Pachter
J. S.
, Liem
R. K. H.
(1984
). The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve.
Dev. Biol
103
, 200
–210
Pannese
E.
, Procacci
P.
, Ledda
M.
, Arcidiacono
G.
, Frattola
D.
, Rigamonti
L.
(1986
). Associations between microtubules and mitochondria in myelinated axons of Lacerta muralis. A quantitative analysis.
Cell Tissue Res
245
, 1
–8
Robbins
E.
, Gonatas
N. K.
(1964
). Histochemical and ultrastructural studies on HeLa cell cultures exposed to spindle inhibitors with special reference to the interphase cell.
J. Histochem. Cytochem
12
, 704
–711
Schecket
G.
, Lasek
R. J.
(1980
). Preparation of neurofilament protein from guinea pig peripheral nerve and spinal cord.
J. Neurochem
35
, 1335
–1344
Schmidt
M. L.
, Carden
M. J.
, Lee
V. M.-Y.
, Trojanowski
J. Q.
(1987
). Phosphate depedent and independent neurofilament epitopes in the axonal swellings of patients with motor neuron disease and controls.
Lab. Invest
56
, 282
–294
Scott
D.
, Smith
K. E.
, O'Brien
B.
, Angelides
K. J.
(1985
). Characterization of mammalian neurofilament triplet proteins: subunit stoichiometry and morphology of nature and reconstituted filaments.
J. Biol. Chem
260
, 10736
–10797
Shaw
G.
, Weber
K.
(1982
). Differential expression of neurofilament triplet proteins in brain development.
Nature
298
, 276
–299
Soltys
B. J.
, Gupta
R. S.
(1992
). Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules- a quadruple fluorescence labeling study.
Biochem. Cell Biol
70
, 1174
–1186
Steinert
P. M.
, Roop
D. R.
(1988
). The molecular and cellular biology of intermediate filaments.
Annu. Rev. Biochem
57
, 593
–625
Sternberger
L. A.
, Sternberger
N.
(1983
). Monoclonal antibodies distinguish phosphorylated and non-phosphorylated forms of neurofilaments in situ.
Proc. Nat. Acad. Sci. USA
80
, 6126
–6130
Takeda
S.
, Okabe
S.
, Funakoshi
T.
, Hirokawa
N.
(1994
). Differential dynamics of neurofilament-H proteins and neurofilament-L protein in neurons.
J. Cell Biol
127
, 173
–185
Tascos
N.
, Mourelatos
Z.
, Gonatas
N. K.
(1995
). On the significance and reproducibility of the fragmentation of the Golgi apparatus of motor neurons in human spinal cords.
J. Neuropath. Exp. Neurol
54
, 331
–338
Thyberg
J.
, Moskalewski
S.
(1992
). Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid.
J. Cell Sci
103
, 1167
–1175
Towbin
J.
, Staehelin
T.
, Gordon
J.
(1979
). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Nat. Acad. Sci. USA
76
, 4350
–4354
Troncoso
J. C.
, Hoffman
P. N.
, Griffin
J. W.
, Hess-Kozlow
K. M.
, Price
D.
(1985
). Aluminum intoxication: a disorder of neurofilament transport in motor neurons.
Brain Res
342
, 172
–175
Troncoso
J. C.
, Sternberger
N. H.
, Sternberger
L. A.
, Hoffman
P. N.
, Price
D. L.
(1986
). Immunocytochemical studies of neurofilament antigens in the neurofibrillary pathology induced by aluminum.
Brain Res
364
, 295
–300
Turner
J. R.
, Tartakoff
A. M.
(1990
). The response of the Golgi complex to microtubule alterations: the roles of metabolic energy and membrane traffic in Golgi complex organization.
J. Cell Biol
109
, 2081
–2088
Vickers
J. C.
, Morrison
J. H.
, Friedrich
V. L.
Jr., Elder
G. A.
, Perl
D. P.
, Katz
R. N.
, Lazzarini
R. A.
(1994
). Age-associated and cell-type-specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit.
J. Neurosci
14
, 5603
–5612
Vikstrom
K. L.
, Borisy
G. G.
, Goldman
R. D.
(1989
). Dynamic aspects of intermediate filament networks in BHK-21 cells.
Proc. Nat. Acad. Sci. USA
86
, 549
–553
Vikstrom
K. L.
, Miller
R. K.
, Goldman
R. D.
(1990
). Methods for analyzing the dynamic properties of intermediate filaments.
Meth. Enzymol
196
, 506
–525
Vikstrom
K. L.
, Lim
S.-S.
, Goldman
R. D.
, Borisy
G. G.
(1992
). Steady state dynamics of intermediate filament networks.
J. Cell Biol
118
, 121
–129
Voyvodic
J. T.
(1989
). Target size regulates calibre and myelination of sympathetic axons.
Nature
342
, 430
–433
Wang
E.
, Goldman
R. D.
(1978
). Functions of cytoplasmic fibers inintracellular organelle movements in BHK-21 cells.
J. Cell Biol
79
, 708
–726
Weiss
P. A.
, Mayr
R.
(1971
). Organelles of neuroplasmic (‘axonal’) flow: neurofilaments.
Proc. Nat. Acad. Sci. USA
68
, 846
–850
Weissenfels
N.
, Wachtmann
D.
, Stockem
W.
(1990
). The role of microtubules for the movements of mitochondria in pinacocytes of fresh-water sponges.
Eur. J. Cell Biol
52
, 310
–314
Welsh
W. J.
, Sukan
J. B.
(1985
). Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli and appearance of intranuclear actin filaments in rat fibroblasts after heat shock treatment.
J. Cell Biol
101
, 1198
–1211
Willard
M.
, Simon
C.
(1983
). Modulation of neurofilament transport during the development of rabbit retinal ganglion cells.
Cell
35
, 551
–559
Xu
Z.
, Cork
L. C.
, Griffin
J. W.
, Cleveland
D. W.
(1993
). Increasedexpression of neurofilament subuit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease.
Cell
73
, 23
–33
Yamasaki
H.
, Bennett
G.
, Itakura
C.
, Mizutani
M.
(1992
). Defective expression of neurofilament protein subunits in hereditary hypotrophic axonopathy of quail.
Lab. Invest
66
, 734
–743
Yang
H. Y.
, Lieska
N.
, Goldman
A. E.
, Goldman
R. D.
(1985
). A 300,000-mol-wt intermediate filament associated protein in baby hamster kidney.
J. Cell Biol
100
, 620
–631
Zackroff
R. V.
, Idler
W. W.
, Steinert
P. M.
, Goldman
R. D.
(1982
). In vitro reconstitution of intermediate filaments from mammalian neurofilament triplet polypeptides.
Proc. Nat. Acad. Sci. USA
79
, 754
–757
This content is only available via PDF.
© 1996 by Company of Biologists
1996
-PolarityCFP.png?versionId=4696)
