Neuronal intermediate filaments: new progress on

an old subject

Zuoshang Xu, Dennis L-Y Dong and Don W Cleveland

The Johns Hopkins University School of Medicine, Baltimore, USA

Neurofilaments (NFs) are the major intermediate filaments in most mature neurons. Genetic approaches have now proven that NFs are an essential determinant for radial growth of axons. NF phosphorylation most probably plays an important role in this function. Further, forced over-expression of NF subunits in transgenic mice yields NF misaccumulation in motor neurons and, subsequently, causes motor neuron dysfunction. This has important implications for human motor neuron diseases because similar accumulations are nearly universally found in the early stages of many motor neuron

disorders.

Current Opinion in Neurobiology 1994, 4:655-661

Introduction

Neuronal intermediate (i.e. 10nm) filaments (nIFs) are major constituents of most neurons. Of the five known intermediate filament (IF) classes (defined by sequence homology and the exon-intron structure of the genes encoding each subunit), the three NF subunits (light, medium and heavy: NF-L, NF-M and NF-H), a-in- ternexin and nestin comprise the type IV IF family, whereas a final nIF, peripherin, falls into the type III class. Among these nIFs, NFs are the most abundant ones in large myelinated axons and are the major nIFs in most mature neurons.

Since the initial visualization of NFs by silver staining in the late 19th century, mounting correlative evidence has pointed to the importance of NFs in specifying the dia- meter (caliber) of large myelinated axons, a property that directly affects conduction velocity. As we recount be- low, genetic approaches have now unequivocally proven this hypothesis, confirming an important role for NFs in establishing or maintaining the structure of axons (which for human motor neurons may extend more than a meter in length). Furthermore, abnormal accumulation of NFs has been seen as a common early feature of many mo- tor neuron disorders [l]. To this background, recent ef- forts have documented that in oivo NFs are obligate het- eropolymers (contrary to earlier in vitro studies) and are surprisingly dynamic. A flurry of reports have also iden- tified a set of kinases that may be involved in multiply phosphorylating the NF-H tail, while companion efforts have indicated that such phosphorylation may affect the ability of NF to mediate radial growth. O-linked gly-

cosylation, a modification found on many cytoplasmic proteins, has been found on multiple sites of all three NF subunits, and may affect assembly properties. Finally, use of transgenic mouse models has provided evidence directly linking NF abnormalities to neurodegenerative processes, particularly in motor neurons.

For recent reviews with a more detailed focus on assem- bly properties [2**], cell biology [3**] or phosphorylation of NFs [4”], we refer readers elsewhere.

Regulation of nlF expression

Expression of nlFs is predominantly neuron specific With few exceptions, nIFs that maintain their expression in mature neurons (the three NF subunits, a-internexin and peripherin) are expressed specifically in neurons. The NF subunits and a-internexin are present in the vast majority of neurons in both the CNS and PNS. Pe- ripherin, however, is detected predominantly in neuronal groups that contact peripheral tissue. The expression of different types of nIFs is neither mutually exclusive nor entirely overlapping. On the one hand, NF subunits are found to co-exist with a-internexin in the CNS [5*] and with peripherin in the PNS [6,7]. On the other hand, distinct neuronal groups that predominantly express ei- ther one or the other nIF have been shown. In peripheral sensory and autonomic ganglia, the largest neurons ex- press NFs and the smallest neurons express peripherin, with somewhat medium-sized neurons expressing both [6,7]. In the same groups of neurons, this expression pat-

Abbreviations A&-amyotrophic lateral sclerosis; bp-basepairs; CDC-cell division cycle; CDK-cyclin-dependent kinase;

CNCentral nervous system; FALS-familial ALS; IF-intermediate filament; NF-neurofilament; NFAK-neurofilament-associated kinase; nlf-neuronal intermediate filament; PNS-peripheral nervous system; SODl-superoxide dismutase 1.

0 Current Biology Ltd ISSN 0959-4388 655

656 Neuronal and giiai cell biology

tern has also been reported for NFs and a-internexin [8]. Another case is in rat cerebellum, where a-internexin is expressed, whereas NFs are undetectable in parallel fibers [5*]. The generality of this finding, however, will require further study as a-internexin has not been detected in human cerebellar parallel fibers [9*].

Although NFs are widely taken to be neuron specific, recent evidence has indicated that NF-M and NF-L may be expressed in immature Schwann cells [lO,l 11, and NF-H in T-lymphocytes [12] and embryonic heart muscles [13] (which also express peripherin [14’]). The significance of this non-neuronal expression is unclear.

Expression of nlFs follows neuronal differentiation and increases as neurons mature The earliest IF associated with neuronal development is nestin, which is expressed at high levels in neuroecto- derm cells that have the potential to develop into both neurons and glia (see [15] and citations therein). Nestin levels begin to decline during cell migration from the ventricular zone to the marginal zone, and vimentin ex- pression initiates. This is followed by another switch, with the silencing of vimentin after terminal differen- tiation of neurons, and induction of nIFs [9*].

The precise sequence of appearance of different nIFs (e.g. in the case of NFs and peripherin) may be slightly different in different species [16,17]. However, common to all nIFs is their appearance soon after terminal neuro- nal differentiation, followed by a gradual increase during late embryonic and early post-natal development [5*,9*]. This process is most dramatic for NFs, which increase rapidly and markedly during post-natal development. An exception to this general trend is expression of periph- erin in motor neurons and large peripheral sensory neu- rons: peripherin increases transiently during embryonic stages but disappears as these neurons mature [17]. Inter- estingly, recurrence of this embryonic expression pattern of nIFs has been observed in adult animals during recov- ery from peripheral axonal injury [15,18,19].

What are the regulatory elements for nlF expression? The pursuit of the regulatory mechanism that specifies high-level expression of NFs in a wide range of neu- rons has been stalled by the difficulty in defining the sequences that reproduce the pattern of NF expression in test genes. Efforts to use cultured cells as a model sys- tem to identity the promoter segment(s) responsible for high neuronal expression have invariably been unsuc- cessful because transfected NF genes express not only in cells of neuronal origin, but equally well in non- neuronal cells, even when long 5’-flanking sequences are included in the constructs [20,21]. Specific neuro- nal expression of NF-L and NF-H, however, has been found in transgenic mice using constructs that include long 5’-flanking sequences, as well as the coding se- quences, introns and 3’-flanking regions [21,22**]. The

most detailed analysis of a NF promoter in transgenic mice has been carried out on the NF-L gene [21,23*].

High-level expression in the nervous system has been achieved with constructs that include 300 basepairs (bp) before the transcription initiation site followed by the rest of the gene (coding sequences and introns) except for the 3’-flanking sequences [21]. Specific expression requires a combined presence of the segment between 300 bp and 190 bp before the transcription initiation site and the intragenic transcribed sequences; exclud- ing either results in ectopic expression [2 1,23*]. A similar requirement also exists for the regulation of peripherin expression, where only the presence of both 5’-flanking and intragenic sequences replicates the expression pat- tern of the endogenous gene [14*].

In addition to transcriptional control, the level of NFs may also be regulated by post-transcriptional mecha- nisms. Julien and colleagues [24*] have shown that in their transgenic mice carrying a human NF-L trans- gene, a three- to five-fold increase in NF-L mRNA corresponds to only a lO-50% increase in NF-L pro- tein in the CNS. Another example is in a mutant strain of quail, where a point mutation that creates a prema- ture stop codon in the NFL gene (truncating 80% of the carboxyl terminus polypeptide sequence) results in significant reduction in both NF-L and NF-H mR- NAs, albeit the former is reduced to a greater extent [25**]. Furthermore, although the NF-M mRNA level is nearly normal, accumulation of the NF-M polypeptide is minimal.

Post-translational modifications of nlFs

The most studied post-translational modification of nIFs is phosphorylation of the NF subunits. Early studies showed that NF subunits are heavily phosphorylated, with most of the phosphates added to the tail domains of NF-H and NF-M and a few in the head domains (summarized in Fig. 1). Phosphorylation on the head domain occurs early after synthesis and some of these sites are turned over quickly during axonal transport. By analogy to the other IFS, these sites may regulate filament assembly. Extensive phosphorylation only ini- tiates when NFs enter axons and continues throughout axonal transport (reviewed in detail in [4**]). Most, if not all, of these phosphates are added onto the serine residues within the repetitive lysine-serine-proline (KSP) motif in NF-M and NF-H [26,27*] and are turned over rela- tively slowly [4**]. Although an unequivocal demonstra- tion for function of NF phosphorylation has not been achieved, significant progress has been made in recent years (see below).

The mechanism by which large number of phosphates are added to NF subunits in axons is an intriguing prob- lem but has yet to be understood. In vitro, NF subunits have been reported as a substrate for a long list of kinases

Neuronal intermediate filaments Xu, Dong and Cleveland 657

o-f’cNAc + 0-ClcNAc? -+

- -25-50 PO, -

NF-H

1 99 274 291 411 519 886 1072

NF-M

1 104 244267 411 849

NF-L

234257 401 543 0 1994 Current Opinmn in Neurobiology

Fig. 1. Polypeptide structure and post-translational modifications of mouse NF proteins. Clear bars represent non-a-helical regions; shaded bars represent a-helical regions. Multiple phosphorylation sites are indicated directly on NF-H [27*] and by arrow heads on NF-M (serine residues 502, 506, 536, 603, 608 and 666) [26] and NF-L (serine residues 55 and 473) (see references in [4**,26]). Mul- tiple phosphorylation sites also exist on the head domain of NF-M, although the exact locations have not been identified. The symbols ( ) indicate the location of glycosylation sites on NF-M fthreonine residues 19,48 and 431; serine 34) and NF-L tthreonine 21; serine residues 27 and 48) [36**]. Clycosylation (O-GlcNAc: N-acetylglu- cosamine linked to serine or threonine) also occurs on the head and tail of NF-H, but the exact location has not been determined.

including A kinase, C kinase [4**], Cap+- calmodulin de- pendent kinase [28], casein kinase I [29*], PK36, PK40 [30], CDC2 kinase [31], CDK5 [32*,33-l, tau kinase II [34*] and NFAK115 [35*]. Among these, the first four and NFAK115 are associated with NFs in vitro; six (A ki- nase, PK36, PK40, CDC2 kinase, CDK5 and tau kinase II) have been shown to phosphorylate the amino acid residues that are also phosphorylated in viva [26,27*]. Phosphorylation by A kinase occurs on some of the residues in the amino-terminal domains of NF-M and NF-L (Fig. 1) [4**] an d cause disassembly of the reassem- bled NFs in vitro [36**]. However, phosphorylation of the native NFs by A kinase results in fewer phophates being incorporated into NF subunits than the reassembled NFs and only induces thinning and partial fragmentation of the filaments, suggesting that the structure of the in vitro reassembled NFs is different from the native NFs [36**]. This is consistent with the recent cell transfection exper- iments indicating differences between assembly in vitro and in viva [3**].

PK36, PK40, CDC2 kinase, CDK5 and tau kinase II all are capable of phosphorylating serines within the KSP repeating motif. Curiously, only phosphorylation by CDC2 kinase and tau kinase II restores the retarded migration of in uivo phosphorylated NF-H, converting the rapidly migrating, dephosphorylated form to the slow moving, fully phosphorylated form on sodium do- decyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) [31,34*]. PK40 is capable of doing the same for NF-M [30]. The sites phosphorylated by casein kinase I and NFAKl15 are likely to be located in the carboxvl terminal tail domain. but the exact residues

have not been determined [29*,35-l. All of these ki- nases are present in brain at abundant levels, but none has been demonstrated to phosphorylate NF subunits in vivo.

Recently, an additional post-translational modification, 0-glycosylation, has been identified on all three NF subunits [37**]. This modification, in which single N- acetylglucosamine monosaccharides are 0-glycosidically linked to serine or threonine residues, is an intracellular form of glycosylation found on many cytoplasmic and nuclear proteins. Precise positions of some of these gly- cosylation sites have been identified (Fig. 1). The dis- covery of 0-glycosylation on NFs raises many inter- esting questions about the function and metabolism of the glycosylation process, including the possibility that glycosylation affects assembly [3**,37**].

Axonal transport of neurofilaments

nIFs are transported along axons in a slowly transported protein group via slow axonal transport, which moves at a rate 0.25-3 mm day-t. The exact transport velocity measured varies on a number of factors, including the group of the neurons, the age of the animal and the location along the nerve. For example, motor neurons transport NFs at a faster rate than retina ganglion neu- rons [3W]. In the same neuronal group, NFs move along axons more rapidly in young animals than old animals [38**], and in proximal axons than distal axons [39].

The mechanism by which NFs are transported remains a matter of speculation. However, several recent re- ports have shed new light on this question. Lasek and colleagues [38**] have observed that some NF subunits move along axons at a rate as fast as 72-144mm day-1 over a short distance. From this, they suggested that NFs may be transported in translocating steps, alternating be- tween short, but fast-moving strides and relatively long pauses. Taking advantage of delayed degeneration upon transection of sciatic nerves in mice with the Ola mu- tation (a spontaneous mutation in an as-yet-unidentified gene), Griffin and colleagues [40**] observed that NFs, as well as other cytoskeletal proteins, redistribute to- wards both distal and proximal transected ends.- These investigators pointed out that this could be caused by a retrograde mechanism of slow axonal transport that may exist in normal axons. A point that remains to be demonstrated is whether axonal organization in Ola mice represents a simple loss of axonal polarity resulting from, for example, changes in microtubule orientation

’ following transection.

A subject of contentious debate with regard to the axonal transport of NFs has been whether NFs are transported together, as one pool at one velocity [41], or separately, as two pools at two velocities: one relatively fast, at the traditionally measured slow axonal transport rate, and the other much slower. so as to be stationarv 1421. Although , ,L >

658 Neuronal and glial cell biology

advocates on both sides acknowledge that in addition to the NFs traveling at a speed of -0.7mm day-l, some NFS travel at a much reduced rate (though the estimated rates differ), disagreement remains on the fraction of fil- aments in the slower pool (estimated by one group to be as much as 32% of the total newly synthesized pool [42] and by the other, to be a negligible proportion [41]). Whichever estimate is closer to the actual amount, it is worth bearing in mind that even with a seemingly small deposition from the total slowly transported NF pool to the relatively stationary pool, the steady state stationary pool can reach a significant proportion of the total given the very slow rate of transport of the stationary pool. For example, if the deposition of NFs is a mere 3% from the total slowly transported pool and if the difference in transport rate is 25 fold (0.75 mm day-’ versus 0.03 mm day-l), at steady state, the stationary pool could reach a significant 44% of the total NFs transported through the axon.

New evidence horn Hirokawa and colleagues [43**] sup- ports the two pool hypothesis. These authors labeled the NF network in neurites of cultured neurons with fluo- rescently tagged NF-L, and then bleached a segment of fluorescence. They observed that the bleached segment recovered gradually in fluorescence but did not move, clearly suggesting that the bulk of the NF network is relatively stationary (there must be a moving pool, of course, or the fluorescence could not recover) [43*-l. In a second experiment, these authors injected biotinylated NF-L subunits into the cell body [430~]. After various periods of time, the filament network in neurites was ex- amined by electron microscopy. The biotinylated NF-L was initially incorporated into existing filaments (in neu- rites 200-300 pm away from the cell body) through lo- calized regions (referred to as subunit incorporation hot spots) on many filaments. At longer time intervals, the labeled NF-L became relatively evenly distributed along the entire filaments [43”]. This result is consistent with a mechanism in which the transported subunits con- stantly exchange with the existing stationary NF net- work as they are traveling along a neurite. This exchange could be regulated by the phosphorylation and dephos- phorylation on the amino-terminal residues, possibly by A kinase and an unknown phosphatase [36**].

The function of neurofilaments: usefulness

versus harmfulness

Neurofilaments are required for establishing normal axonal diameter The function of NFs as a major determinant of axonal diameter has long been suspected horn the close cor- relation between the number of filaments in axons and the axonal diameters during all phases of the radial ax-

onal growth that takes place beginning with myelination and continuing through adult life [18]. Definitive proof now has emerged from two animal models. The first is a quail with a spontaneous mutation in NF-L that gen- erates a new termination codon that stops translation prematurely [25**]. The second is a transgenic mouse line that expresses an NF-H subunit with nearly the entire 120 kDa fi-galactosidase polypeptide attached to its carboxyl terminal tail. Remarkably, expression of this mutant NF-H completely stops all NFs from being trans- ported into axons [44”]. Axons from both animals lack NFs completely. As a result, the axonal diameters from both animals are significantly reduced [44**,45*“] and, at least in the quail, there is also, as expected, a significant reduction in axonal conduction velocity [45**].

So how do NFs determine axonal diameter? A critical role for NF phosphorylation has been proposed for many years. According to this proposal, the large number of phosphates added to the tail domain of NF-H and NF- M would increase the interfilament repulsion as a con- sequence of the large number of negative charges. This in turn forces a wider interfilament spacing and, conse- quently, overall axonal caliber expands. This hypothesis has gained new support from some recent experiments where a direct correlation between NF phosphorylation and interfilament spacing in axons has been found. In myelin-deficient trembler mice, NFs are less phospho- rylated and are closely spaced in axons [46]. By com- parison, NFs are, fully phosphorylated and widely spaced in wild-type mice [46]. Further, this correlation remains even in wild-type setting. In the initial segment of dorsal root and retinal ganglion neurons, as well as in nodes of Ranvier, where myelination does not occur, NFs are less phosphorylated and more closely packed than in adjacent myelinated segments [47*,48*]. Statistical analysis of the distribution of NFs in cross sections of myelinated ax- ons also reveals a non-random nature of this distribution: the presence of a minimum filament spacing of - 25 nm between NFs favors repulsive interaction between NFs [49*]. These data also suggest that the phosphorylation process is ultimately linked to, and may be regulated by, myelination.

Fig. 2. Proposed pathway of motor neuron dysfunction and degen- eration in motor neuron disease. The excessive NF burden in motor neurons makes them particularly susceptible to various types of as- sault that disrupt the normal NF transport mechanism.

Neuronal intermediate filaments Xu, Dong and Cleveland 659

Participation of neurofilaments in the pathogenesis of motor neuron disease Severe accumulation of NFs in cell bodies and proxi- mal axons of spinal motor neurons has been observed in many cases of motor neuron disease from both hu- man and animal species [1,50*]. However, until recently, it was not clear whether the accumulation of NFs is an integral part of the pathogenic process that leads to the ultimate neuronal dysfunction and degeneration or if it is simply a harmless by-product of neuronal pathology. Two experiments, both of which used a transgenic ap- proach to over-express NFs (in one case the NF-L subunit [51**] and in the other NF-H [22**]) in mo- tor neurons, have produced mice that highly resemble both the phenotype and pathology of motor neuron dis- ease. These experiments strongly indicate that excessive NF accumulation in cell bodies and proximal axons can lead to neuronal dysmnction and degeneration (Fig. 2). Consistent with this conclusion is the more recent ob- servation that severe NF accumulation in cell bodies of motor neurons (caused by the expression of a mutant NF-H) is accompanied by nearly 25% loss of axons in the ventral roots in 13 month old animals [44**].

The primary causes for NF accumulation in motor neu- ron disease is not entirely clear at this time. Over-pro- duction is unlikely as it has not been observed in animal models of motor neuron disease [52*], nor is it a con- dition necessary for NFs to accumulate [53]. Also less probable is a decrease in degradation, as this would most probably cause NFs to accumulate at the distal end of ax- ons. Such distal accumulation is not observed in motor neuron disease. The most plausible cause for filaments to accumulate in the proximal axons and cell bodies is a defect(s) in slow axonal transport (Fig. 2), which, in turn, could be precipitated by a variety of factors affecting either the slow axonal transport machinery (e.g. trans- porter molecules) or NF structure (e.g. mutation [54”], aggregation [55]).

Evidence is emerging that one of these factors is muta- tions in superoxide dismutase 1 (SODI), which causes - 20% of familial amyotrophic lateral sclerosis (FALS) (-2% of total ALS) [56**]. Precisely how SOD1 mu- tations provoke selective death of motor neurons is un- explained, but a key clue has emerged from the produc- tion of a transgenic mouse model that expresses one of the FALS-linked SOD1 subunits [57**]. Selective lower motor neuron death in these mice is accompanied by masses of NFs in perikarya and proximal axons. Because transgenic mice displaying such accumulations (through merely raising NF synthesis [22**,51**]) develop motor neuron disease in the absence of other defects, the sim- plest view is that SOD1 mutation somehow affects NF accumulation, thereby creating masses of filaments that are destructive to motor neurons. This reasoning is di- rectly applicable to-human ALS, as the spectacular neu- rofilamentous accumulation reported in a detailed ex- amination of one large FALS kindred [58] is caused by mutation in SOD1 (T Siddique, personal communica-

tion). This is further confirmed by a second ALS family with SODI-induced disease; again, aberrant neurofil- amentous aggregates are prominent pathologic features (MB Clark, G Rouleau, personal communication). The idea that NFs play a key pathogenic role offers an attrac- tive explanation for the late onset (age 4&50) of ALS, as NFs naturally increase in abundance during ageing (j Marszalek, Z-S Xu, DW Cleveland, unpublished data).

Conclusion

Two key properties of NFs have now been proven. In the normal context, NF are obligatory for the normal radial growth of axons that occurs concomitant with myelination and is essential for establishment of nor- mal conduction velocity. In the pathologic state, aberrant NF accumulation arising directly as a consequence of in- creased NF expression causes motor neuronal dysfunc- tion. When combined with the discovery that the early pathologic hallmark of ALS is similar neurofilamentous accumulations in both sporadic and SOD1 mutation- induced familial disease, neurof&nentous misaccumu- lation is almost certainly a key pathologic intermediate leading to neuronal failure.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest . . of outstanding interest

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By combining knowledge on the assembly and structure of other IFS and direct experimental data on NFs, the authors summarize and discuss the assembly process and a structural model of NFs.

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in Neurofilament-1 Gene. J Cell Biol 1993, 121:387-395. A premature stop codon in the NF-L gene causes the absence of NFs in a mutant quail whose axons are significantly smaller than wild-type animals. Together with earlier reports describing these mutant quails, this proves that NF accumulation is not essential for survival, but absence of NFs does lead to an overt phenotype of generalized quivering.

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An unequivacal demonstration of phosphorylation in the KSP repeats of NF-H, by means of protein chemistry.

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ment Proteins: Isolation and Characterization. Proc Nat/ Acad SC; USA 1993, 90:6844-6848.

A demonstration that CDKS isolated from brain phosphorylates subsets of KSP repeats in NF-H.

33. Beaudett KN, Lew H, Wang JH: Substrate Specificity Charac- . terization of a cdc2-like Protein Kinase Purified from Bovine

Brain. J Biol Chem 1993, 268:20825-20830. Like 132.1, another report showing the specific phosphorylation of a sub- set of KSP repeats in NF-H by CDKS purified from brain.

34. Hisanaga 5, lshiguro K, Uchida T, Okumura E, Okano T, Kishi- . moto T: Tau Protein Kinase II Has a Similar Characteristic to

cdc2 Kinase for Phosphorylating Neurofilament Proteins. J t?io/ Chem 1993, 268:15056-l 5060.

Tau protein kinase II is shown to phosphorylate dephosphorylated NF- H and shift its apparent molecular weight from the unphosphorylated form to the phosphorylated form. This phosphorylation also diminishes the binding of NF-H to microtubules.

35. Xiao J, Monteiro MJ: Identification and Characterization of a . Novel (115 kDa) Neurofilament-Associated Kinase. J Neurosci

1994, 14:182C-1833. A 115 kDa kinase fNFAK115) isolated from brain is found to associate and phosphorylate NF-H.

36. Hisanaga S, Matsuoka Y, Nihsizawa K, Saito T, lnagaki M, Hi- . . rokawa N: Phosphorylation of Native and Reassembled Neu-

rofilaments C~pos&l of NF-1, NF-M, and NF-H by the Cat- alytic Subunit of cAMP-Dependent Protein Kinase. MO/ Biol Cell 1994, 5:161-172.

Neuronal intermediate filaments Xu, Dona and Cleveland 661

Phosphorylation by A kinase incorporates more phosphates into the in vitro reassembled NFs than the native NFs, and causes disassembly of the former but only thinning and partial fragmentation for the latter.

37. . .

Dong D, Xu Z-S, Chevrier M, Cotter R, Cleveland D, Hart G: ClycosylaGon of Mammalian Neurofilaments. 1 Biol Chem 1993, 268:16679-l 6687.

Using multiple criteria, the authors conclusively demonstrate that NF subunits are modified heavily by a unique intracellular post-transla- tional addition of 0-ClcNAc (N-acetylglucosamine linked to serine or threonine). Many 0-ClcNAc sites that are identified and most are lo- calized at the amino-terminal head domain. Deletions encompassing these sites have previously been shown to have a dominant effect on NF assembly.

38. Lasek RI, Paggi P, Katz MJ: The Maximum Rate of Neurofiia- . . ment Transport in Axons: a View of Molecular Transport Mech-

anisms Continuously Engaged. Brain Res 1993, 61658-64. By observing the wave front of transported NFs in optic nerve shortly after injecting 3%methionine into mouse eye, NFs are shown to move at a much faster rate than slow axonal transport, leading to a new proposal on the mechanism of NF transport.

39. Watson DF, Hoffman PN, Fitto KP, Griffin JW: Neurofilament and Tubulin Transport Slows along the Course of Mature Motor Axons. Brain Res 1989, 477:225-232.

40. Watson DF, Glass JD, Griffin JW: Redistribution of Cytoskeletal . . Proteins in Mammalian Axons Disconnected from Their Cell

Bodies. / Neurosci 1993, 13:4354-4360. Quantitation of NFs in transected nerves of O/a mice indicates an ac- cumulation of NFs in both the distal and proximal transected ends, suggesting the possibility of retrograde axonal transport of NFs.

41. Lasek RJ, Paggi P, Katz MJ: Slow Axonal Transport Mechanisms Move Neurofilaments Relentlessly in Mouse Optic Axons. / Cell Bio/ 1992, 117:607-616.

42. Nixon RA, Logvinenko KB: Multiple Fates of Newly Synthe- sized Neurofilament Proteins: Evidence for a Stationary Neu- rofilament Network Distributed Nonuniformly along Axons of Retinal Ganglion Cell Neurons. 1 Cell Viol 1986, 102:647-659.

43. Okabe S, Miyasaka H, Hirokawa N: Dynamics of the Neuronal . . Intermediate Filaments. j Cell Bio/ 1993, 121:375-386. An important contribution that establishes two points: first, NFs are dy- namic in neurons; second, the bulk of NFs in neurites does not move together as one pool, suggesting a large proportion of NFs is stationary.

44. . .

Eyer J, Peterson A: Neurofilament-Deficient Axons and Perikaryal Aggregates in Viable Transgenic Mice Expressing a Neurofilament-&Galactosidase Fusion Protein. Neuron 1994, 12:389-405.

Expression of a mutant NF-H that has a bgalactosidase sequence at- tached to its carboxyl terminal end totally blocks NF transport into axons, resulting in accumulation of NFs in cell bodies and depletion of NFs in axons. A reduction in the number of axons in the ventral root of old animals suggests that there is a slow degeneration of the motor neurons.

45. . .

Sakaguchi T, Okada M, Kitamura T, Kawasaki K: Reduced Dia- meter and Conduction Velocity of Myelinated Fibers in the Sciatic Nerve of a Neurofilament-Deficient Mutant Quail. Neurosci Len 1993, 153:65-68.

The paper shows that the axons in a mutant quail that lacks NFs are considerably smaller and that the conduction velocity of these axons is significantly reduced.

46. DeWaegh SM, Lee VM-Y, Brady ST: local Modulation of Neurofihment Phosphorylation Axonal Caliber and Slow Ax- onal Transport by Myelinating Schwann Cells. Cell 1992, 68:451-463.

47. Hsieh S-T, Kidd GJ, Crawford TO, Xu Z-S, Trapp BD, Cleveland . DW, Griffin JW: Regional Modulation of Neurofilament Orga-

nization by Myelination in Normal Axons. I Neurosci 1994, in press.

Quantitation of phosphorylation and inter-NF distances in both myeli- nated and non-myelinated axonal segments from dorsal root ganglion neurons indicates a correlation between phosphorylation and wider inter-NF spacing.

48. Nixon RA, Paskevich PA, Sihag RK, Thayer CY: Pbosphorylation . on Carboxyl Terminus Domains of Neurofilament Proteins in

Retinal Ganglion Cell Neurons in Viva: Influences on Regional

Neurofilament Accumulation, Inter-neurofilament Spacing and Axonal Caliber. / Cell Biol 1994, 126:1031-l 046.

Low level of phosphorylation in the initial segment of optic axons (which are not myelinated) correlates with a short inter-NF distances. In contrast, in the more distal optic nerve, where NF are highly phosphorylated, NFs are widely spaced.

49. Hsieh S-T, Crawford TO, Griffin JW: Neurofilament Distribution . and Organization in the Myelinated Axons of the Peripheral

Nervous System. Brain Res 1994, 642:316-326. A statistical approach evaluating the pros and cons of various methods used in analyzing NF distribution in axons. The study shows non-ran- domness in NF distribution, which is consistent with a repulsive interac- tion between NFs.

50. Xu Z-S, Cork LC, Griffin JW, Cleveland DW: Involvement of . Neurofihments in Motor Neuron Disease. I Cell SC; 1993,

106(suppl 17):101-108. The review summarizes current evidence that NF pathology is involved in both human as well as spontaneous animal motor neuron disease.

51. Xu Z, Cork L, Griffin J, Cleveland D: Increased Expression of . . Neurofilament NF-1 Produces Morphological Alterations that

Resemble the Pathology of Human Motor Neuron Disease. Cell 1993, 73:23-33.

Transgenic mice over-expressing NF-L display phenotypic and pathologic features of motor neuron disease. This finding, in conjunction with those of [22**1, strongly implicate abnormal NF accumulation as a key patho- logica intermediate in human disease.

52. Muma NA, Cork LC: Alterations in Neurofilament mRNA in . Hereditary Canine Spinal Muscular Atrophy. Lab invest 1993,

69436-442. By in situ hybridization, expression of NFs in motor neurons from dogs with hereditary spinal muscular atrophy is found to be reduced, despite the massive NF accumulation in these neurons during the course of the disease.

53. Muma NA, Troncoso JC, Hoffman PN, Koo EH, Price DL: Aluminum Neurotoxicity: Altered Expression of Cytoskeletal Genes. Brain Res 1988, 427:115-l 21.

54. . .

Figlewicz DA, Krizus A, Martinol MC, Meininger V, Dib M, Rouleau GA, Julien J-P: Variants of the Human Heavy Neu- rofilament Subunit Are Associated with the Development of Amyotrophic Lateral Sclerosis. Human MO/ Genet 1994, in press.

Five mutations (deletion of a single lysine or a 34 amino acids in the KSP repeats) in the carboxyl terminus domain of NF-H are found in 356 sporadic ALS patients. None of these mutations is found after screen- ing 306 normal controls. The authors suggest that these mutations might predispose the patients to developing ALS.

55. Troncoso JC, March JL, Haner M, Aebi U: Effect of Aluminum and Other Multivalent Cations on Neurofilameds in W&a an Electron Microscopic Study. / Srrucf Biol 1990, 103:2-l 2.

56. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, . . Hentati A, Donaldson D, Goto J, Oregan JP, Deng HX et al.:

Mutations in Cu/Zn Superoxide Dismutase Gene are Associ- ated with Familial Amyotrophic Lateral Sclerosis. Nature 1993, 362:59-62.

Eleven different missense mutations in the human SOD1 gene are iden- tified to cause FALS.

57. Gurney ME, Pu HF, Chiu AY, Dalcanto MC, Polchow CY, . . Alexander DD, Caliendo J, Hentati A, Kwon YW, Deng HX

et a/.: Motor Neuron Degeneration in Mice That Express a Human Cu/Zn Superoxide Dismutase Mutation. Science 1994, 264:1772-l 775.

Expression of a mutant SODl subunit that is known to cause ALS in hu- mans results in severe accumulation of NFs in motor neurons and motor neuron degeneration.

~58. Hirano A, Nakano I, Kurland LT, Mulder DW, Halley PW, SC- comanno G: Fine Structural Study of Neurofibrillary Changes in a Family with Amyotrophic Lateral Sclerosis. 1 Neuropathol Exp Neural 1984, 43:471-480.

Z Xu, DL-Y Dong and DW Cleveland, Department ofBiobgical Chemistry, The Johns Hopkins University School of Medicine, 735 North Wolfe Street, Ualtimore, Maryland 31705, USA.