, Vol. 84, 663–666, March 8, 1996, Copyright 1996 by Cell Press Neuronal Growth and Death: Minireview Order and Disorder in the Axoplasm

Don W. Cleveland no in as a consequence of prema- Ludwig Institute for Cancer Research ture translation termination in NF-L (Ohara et al., 1993). and Departments of Medicine and This results in no detectable NF-L , total absence University of California, San Diego of neurofilaments, and resultant axons that almost com- 9500 Gilman Drive pletely fail to grow radially. La Jolla, California 92093 A central limit to neurofilment-dependent radial growth is the velocity and quantity of components deliv- ered to axons by . Transport is a neces- , whose long thin axonal processes represent sity for neurons, as protein synthesis is restricted to the conduits for electrical signaling, are the most asym- cell bodies and . Membrane-bound particles metric cells in nature. Asymmetry arises in two steps, in axons are trafficked rapidly in both directions using each mediated by different cytoskeletal elements. Initial ATP-dependent motors. The remaining neurite elongation utilizes actin/myosin for growth cone components, including all known cytoskeletal , locomotion and as tracks along which pro- teins and membranes are delivered from the cell body toward the developing terminus. After a stable has formed, a second phase, termed radial growth, is initiated during which neurofilaments, the in- termediate filaments of most large neurons, accumulate to become the most abundant cytoskeletal elements (Figure 1B) and the axonal diameters increase by up to an order of magnitude (leading to up to a 100-fold in- crease in volume!). Radial growth initially occurs rapidly, concomitant with myelination, and then continues slowly throughout adulthood. This is important to neu- ronal function since diameter represents one of the two key determinants specifying velocity of electrical signal conduction, the other being the amount of insulation provided by . For the meter-long human motor and sensory neurons of the peripheral , the initial elongation phase yields a cell >104 times longer than most animal cells, while radial growth yields a cell volume 103 times bigger than is typical and >99.9% of which is in the axon (see Figure 1A). These properties highlight a key general question in cell biology: what are the principles speci- fying cell volume and how does a structural scaffolding, such as an array of neurofilaments (Figure 1B), deter- mine and maintain cell (or in the present examples, axo- nal) volume? What I attempt here is review of what is known about how neurofilaments mediate radial growth and how aberrations in filament organization can be direct causes of selective motor death that mim- ics the most frequent human disease amyotrophic lateral sclerosis (ALS). Neurofilaments as Determinants of Radial Growth Since the initial visualization of neurofilaments by the Figure 1. Structure of Axoplasm great neuroanatomists of the 19th century, mounting (A) Schematic representation of the structural features and asymme- evidence has pointed to their importance as primary try of a human motor neuron. The enlarged portion shows neurofila- -␮m diameter segments (internodes) myelin 10ف determinants of radial growth in myelinated axons. ment arrays in the ated by single Schwann cells and in the 1 ␮m diameter unmyelinated Early correlative studies showed a linear relationship segments (nodes). Phosphorylation of tail domains of NF-M and NF- between number and cross-sectional H is restricted to myelinated segments. area throughout normal radial growth and during re- (B) Quick freeze, deep etch view of the of a myelinated growth following axonal injury. That neurofilaments, as- axon from rat sciatic . A single (25 nm) microtubule, with two sembled from NF-L (68 kDa), NF-M (95 kDa), and NF-H vesicles that are probably moving along it, is seen in the center; all .nm) are neurofilaments. Bar, 100 ␮m 10ف) kDa), are a primary influence, and not a conse- the remaining filaments 110) (Micrograph kindly provided by N. Hirokawa.) quence, of the caliber of myelinated axons has been (C) Rotary shadowed view of a native neurofilament showing the proven unequivocally with the identification of a mutant tail domains of NF-M and NF-H extending from the core of the Japanese quail (named quiver or Quv) that accumulates filament. Bar, 100 ␮m. (Micrograph kindly provided by U. Aebi.) Cell 664

are moved in a phase called slow transport (at a rate of 1992). Furthermore, convergence of evidence from sev- a few millimeters per day) using motor(s) which have eral directions makes it seem likely that phosphorylation not been identified. Also unresolved for cytoskeletal of the NF-M tail may determine the minimal interfilament components is whether polymers, oligomers, or mono- distance (but as discussed above, this is not sufficient mers are transported. What is clear is that the burden for radial growth). In the myelinated portions of axons of neurofilaments has a direct effect on the rate of slow (the internodes), where NF-M is fully phosphorylated, nm, while in the 50ف transport. Increases in NF-H, achieved by expressing the filament–filament spacing is wild type human NF-H in mice at a level about twice the unmyelinated portions of axons (see enlarged portion endogenous NF-H (Coˆ te´ et al., 1993), selectively reduce of Figure 1A) and between the nonphosphorylated fila- transport velocity of neurofilaments (Collard et al., 1995), ments assembled following expression of NF-L and 30ف with transport of other components more modestly af- NF-M in nonneuronal cells, the spacing drops to fected. This in turn alters caliber in two ways: proximal nm (Nakagawa et al., 1995). axons have increased neurofilament content and larger The signaling pathway through which the myelinating diameters, while distal axons atrophy (Coˆte´ et al., 1993). cells communicate to axons to stimulate phosphoryla- How do neurofilaments structure axoplasm so as to tion is not known. Several kinases have been implicated, mediate caliber? The linear correlation between neurofi- but none has yet been shown to reproduce the phos- lament number and axonal cross-sectional area initially phorylation found in myelinated segments. suggested that the axon expanded or contracted in or- Neurofilaments and Motor Neuron Disease der to maintain a constant density of neurofilaments Beyond their role in growth and maintenance of axonal (albeit different densities were found in different nerve caliber, three lines of evidence have combined to impli- types). Raising the filament number by elevating wild cate aberrant neurofilament accumulation in the patho- type NF-L in transgenic mice has disproven this simple genesis of motor neuron disease, including the one most view. Both the number and density of neurofilaments is abundant in humans, amyotrophic lateral sclerosis, or increased two- to three-fold, but this inhibits radial ALS, onset of which occurs in midlife (40–50 years of growth (Monteiro et al., 1990). Thus, while NFs are re- age). The classic hallmark of ALS and most other motor quired for normal radial growth of axons, determination neuron diseases is selective failure, degeneration, and of axonal diameter by NF is specified by factors in addi- death of motor neurons, which in turn triggers skeletal tion to simple filament number. muscle atrophy, almost invariably culminating in paraly- One reasonable postulate is that a volume determin- sis and death. That aberrant accumulation of neurofila- ing, three dimensional array of filaments arises from ments might play a role in disease progression was interactions involving the tail domains of the NF-M and first suggested by the discovery that accumulation and NF-H subunits, which extend from the surface of the abnormal assembly of neurofilaments are common filaments (Figure 1C). In addition to the characteristic pathologic hallmarks found in the early stages of dis- amino acid helical segment required for 310ف central ease. More direct evidence emerged from expression assembly, an obvious feature of the NF-M and NF-H subunits is the length and sequences of the carboxy- in transgenic mice of three to four times the normal terminal tail regions, which in the case of NF-H contains levels of wildtype murine NF-L (Xu et al., 1993) or wild- type human NF-H (Coˆ te´ et al., 1993). Both of these pro- -amino acids comprised primarily of a motif re 850ف peated 40–50 times and containing a central KSP tripep- duced progressive loss of kinetic activity, muscle atro- tide, the serine of which is nearly stoichiometrically phy, and paralysis. This was accompanied by large phosphorylated in myelinated axonal segments. A test accumulations of closely packed neurofilaments both of how subunit composition, rather than filament num- in distended motor neuron cell bodies and in proximal ber, affects radial growth, has been provided by axonal swellings, albeit in neither instance was there transgenic mice that express about twice the normal significant neuronal death even in the oldest animals level of NF-M in motor and sensory neurons (Wong et (Xu et al., 1993; Collard et al., 1995). Proof that mutations al., 1995a). Increases in NF-M are accompanied by de- in neurofilaments can be primary causes of motor neu- creases in NF-H content and a corresponding inhibition ron disease emerged from expression in mice of modest of radial growth, but the total molar content of neurofila- levels of a point mutant in NF-L. In multiple transgenic ment subunits and the spacing between adjacent fila- lines, this caused not only progressive motor neuron ments (i.e., the nearest neighbor distances) remain disease with its accompanying skeletal muscle atrophy unchanged. The simplest hypothesis is that NF-H-de- and abnormal accumulations of neurofilaments that pendent interactions or crossbridging between nonadja- closely mimicked those seen in ALS, but also selectively cent neurofilments or between neurofilaments and other killed lower motor neurons (Lee et al., 1994). As in human axonal components (including microtubules and cortical disease, the large motor axons with abundant neurofila- actin arrays) play crucial features in radial growth. ments were lost, while smaller axons (with fewer neuro- Strong support for the involvement of the highly phos- filaments) were spared. Also as in ALS, the largest (neu- phorylated NF-M and NF-H tail domains in mediating rofilament-rich) sensory neurons were also killed. axonal organization has emerged from examination of These findings have raised the real possibility that the Trembler mouse. Myelination fails in these mice as damage to neurofilaments, including mutations in neu- the result of mutation in the gene encoding myelin basic rofilament genes, may be a primary cause of ALS or protein. The absence of normal myelination triggers a other human motor neuron diseases. Further evidence cascade of events that results in decreased phosphory- for mutations in neurofilaments as primary or contribu- lation of NF-H and NF-M, increase in neurofilament den- tory factors in ALS has come from examination of the sity and inhibition of radial growth (de Waegh et al., KSP repeat domain of NF-H in DNAs from 356 sporadic Minireview 665

ALS patients: this has uncovered 1 or 34 codon deletions in 5 patients, but not in over 300 normal individuals (Figlewicz et al., 1994). Whether such neurofilament mu- tations appear in dominantly inherited familial disease is not yet reported. If neurofilamentous accumulations are important in- termediates in human disease, how can this be recon- ciled with the discovery (reviewed by Brown, 1995) that of total ALS cases) result %2ف) of familial ALS %20ف from mutations in the abundant, ubiquitously expressed intracellular enzyme superoxide dismutase (SOD1)? Three observations are relevant here: First, for three familial ALS-linked SOD1 mutations, the corresponding pathology in patients is known and in all cases neurofilamentous accumulations in cell bodies and proximal axons are a conspicuous feature. This is especially true for the most frequent SOD1 mutation in Figure 2. Model of Motor Neuron Degeneration and Death Caused familial ALS (alanine substituted for valine at amino acid by Blockage of Axonal Transport 4, or A4V); detailed examination of several patients has See text for details. shown neurofilament accumulation to be pronounced (Hirano et al., 1984). Second, expression in mice of two different SOD1 Neuronal degeneration and death is a long, lingering mutants (Gurney et al., 1994; Wong et al., 1995b), but not process that ensues from a no more sophisticated of wildtype human SOD1, produces progressive motor mechanism than strangulation of the axon (depicted in neuron disease characterized by large axonal swellings Figure 2). Combined with the naturally occurring, age- containing either aberrant masses of neurofilaments or dependent increase in neurofilaments per axon, disor- membrane-bound structures that appear to represent dered axoplasm arising from damaged neurofilament degenerating mitochondria (Dal Canto and Gurney, arrays offer at least a partial explanation for the late 1994; Wong et al., 1995b). onset of disease and the selective vulnerability of large Third, disease in these SOD1 mutant–expressing mice diameter, neurofilament-rich motor neurons. arises not from diminution (or elevation) of SOD1 activity (which converts superoxide into hydrogen peroxide) but rather from an as yet unidentified toxic property or prop- Selected Reading erties of the mutant subunits (Gurney et al., 1994; Wong Beckman, J.S., Carson, M., Smith, C.D., and Koppenol, W. H. (1993). et al., 1995b). Nature 364, 584. Two proposals have been offered for the toxicity of the SOD1 mutants. In the first, the mutant SOD1 causes Brown, R.H., Jr. (1995). Cell 80, 687–692. nitration of tyrosine residues on proteins by using Collard, J.F., Coˆ te´ , F., and Julien, J.P. (1995). Nature 375, 61–64. peroxynitrite, formed by spontaneous reaction of super- Coˆ te´ , F., Collard, J.F., and Julien, J.P. (1993). Cell 73, 35–46. oxide with nitric oxide, as a substrate (Beckman et al., Dal Canto, M.C., and Gurney, M.E. (1994). Am. J. Pathol. 145, 1271– 1993). In the second (Wiedau-Pazos et al., 1996), the 1279. mutant enzymes work aberrantly as peroxidases, cata- de Waegh, S.M., Lee, V.M., and Brady, S.T. (1992). Cell 68, 451–463. lyzing oxidation of substrates using the normal end product hydrogen peroxide. Consistent with this idea, Figlewicz, D.A., Krizus, A., Martinoli, M.G., Meininger, V., Dib, M., four SOD1 mutants have been shown in vitro to catalyze Rouleau, G.A., and Julien, J.-P. (1994). Human Mol. 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Neuron 13, 975–988. neurofilaments and other slowly transported proteins require transit times from synthesis to arrival near the Monteiro, M.J., Hoffman, P.N., Gearhart, J.D., and Cleveland, D.W. nerve terminus of one to two years for the meter long (1990). J. Cell Biol. 111, 1543–1557. axons populating the human sciatic nerve (assuming Nakagawa, T., Chen, J., Zhang, Z., Kanai, Y., and Hirokawa, N. that the transport rate in humans is similar to that mea- (1995). J. Cell Biol. 129, 411–429. sured in rodents [0.5–2 mm/day]). In this freeway acci- Ohara, O., Gahara, Y., Miyake, T., Teraoka, H., and Kitamura, T. dent model of neuronal death, damaged proteins slowly (1993). J. Cell Biol. 121, 387–395. accumulate, leading to disordered axonal filament Wiedau-Pazos, M., Goto, J.J., Rabizadeh, S., Gralla, E.B., Roe, J.A., arrays and a gradual poisioning of further transport. Valentine, J.S. and Bredesen, D.E. (1996). Science, in press. Cell 666

Wong, P.C., Marszalek,J., Crawford, T.O., Xu, Z., Hsieh, S.-T.,Griffin, J.W., and Cleveland, D.W. (1995a). J. Cell Biol. 130, 1413–1422. Wong, P.C., Pardo, C.A., Borchelt, D.R., Lee, M.K., Copeland, N.G., Jenkins, N.A., Sisodia, S. S., and Price, D.L. (1995b). Neuron 14, 1105–1116. Xu, Z-S., Cork, L.C., Griffin, J.W., and Cleveland, D.W. (1993). Cell 73, 23–33.