Human midsized neurofilament subunit induces motor neuron disease in transgenic mice

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Experimental Neurology 184 (2003) 408 – 419

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Human midsized neurofilament subunit induces motor neuron disease in transgenic mice Miguel A. Gama Sosa,a,1 Victor L. Friedrich Jr.,b,1 Rita DeGasperi,a,1 Kevin Kelley,c Paul H. Wen,a Emir Senturk,a Robert A. Lazzarini,d and Gregory A. Eldera,* a

Department of Psychiatry, Mount Sinai School of Medicine, New York, NY 10029, USA The Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029, USA c The Mouse Genetics Shared Resource Facility, Mount Sinai School of Medicine, New York, NY 10029, USA d Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029, USA b

Received 3 January 2003; revised 25 March 2003; accepted 26 March 2003

Abstract Aberrant accumulation of neurofilaments is a feature of human motor neuron diseases. Experimentally motor neuron disease can be induced in transgenic mice by overexpressing the mouse neurofilament light subunit (NF-L), the human heavy subunit (NF-H), or mouse peripherin. Here we describe that mice harboring a bacterial artificial chromosome (BAC) transgene containing the human midsized neurofilament subunit (NF-M) gene develop a progressive hind limb paralysis associated with neurofilamentous accumulations in ventral horn motor neurons and axonal loss in ventral motor roots. Biochemical studies revealed that all three mouse neurofilament subunits along with the human NF-M contributed to filament formation, although filaments contained less peripherin. In addition the endogenous mouse NF-M became less phosphorylated in the presence of the human protein and accumulated in the cell bodies of affected neurons even though phosphorylated human NF-M did not. Remaining motor axons contained an increased density of neurofilaments and morphometric studies showed that principally small myelinated axons were lost in the transgenic animals. Removing half of the mouse NF-M by breeding the transgene onto the mouse NF-M heterozygous null background offered no protection against the development of disease, arguing that the effect is not simply due to elevation of total NF-M. Collectively these studies argue that the human and mouse NF-M proteins exhibit distinct biochemical properties and within mouse neurons are not interchangeable and that indeed the human protein may be toxic to some mouse neurons. These studies have implications for the use of human neurofilament transgenic mice as models of amyotrophic lateral sclerosis. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Anterior horn cells; Cytoskeletal proteins; Intermediate filaments; Midsized neurofilament subunit; Neurofilaments; Transgenic mice; Motor neuron disease

Introduction Neurofilaments (NFs) form by polymerization of three subunits termed the light (NF-L), midsized (NF-M) and heavy (NF-H) NF proteins. In mammals separate genes code for each subunit (Julien et al., 1986; Lees et al., 1988; Myers et al., 1987) which share common structural features

* Corresponding author. Department of Psychiatry/Box 1229, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA. Fax: ⫹1-212-860-9279. E-mail address: [email protected] (G.A. Elder). 1 These authors contributed equally to this work.

with other members of the intermediate filament family (Lee and Cleveland, 1996). In myelinated axons NFs are the most abundant cytoskeletal elements and null mutations of each of the NF genes have shown that functionally NFs are central determinants of axonal diameter (Elder et al., 1998a, 1998b; Rao et al., 1998; Zhu et al., 1997, 1998). Aberrant accumulation of NFs is a feature of many neurodegenerative diseases including human motor neuron diseases such as amyotrophic lateral sclerosis (ALS) (Julien and Beaulieu, 2000). About 10% of ALS cases are inherited in an autosomal dominant fashion with mutations in the superoxide dismutase 1 (SOD1) gene accounting for ⬇20% of these cases. The etiology of the remaining familial cases

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M.A. Gama Sosa et al. / Experimental Neurology 184 (2003) 408 – 419

and the much larger number of sporadic cases is unknown. NFs accumulate in anterior horn cells early in the disease course, leading to speculation that the accumulations might actually cause motor neuron dysfunction. Support for this idea has come from studies in transgenic mice showing that overexpression of mouse NF-L (Xu et al., 1993), a mouse NF-L rod domain mutant (Lee et al., 1994), human NF-H (Cote et al., 1993), or mouse peripherin (Beaulieu et al., 1999) can produce motor neuron disease. In addition overexpression of mouse NF-M (Wong et al., 1995), mouse NF-H (Marszalek et al., 1996), or a mouse NF-H/LacZ fusion protein (Eyer and Peterson, 1994) can cause massive accumulations of NFs in ventral horn motor neurons although no overt motor dysfunction. Previous transgenic mice generated with an 8.5-kb genomic fragment containing the human NF-M gene (Lee et al., 1992) developed age-dependent accumulations of NFs in neocortex (Vickers et al., 1994) but never developed motor symptoms or motor neuron pathology. Collectively these studies could be seen as suggesting that mouse motor neurons may be less sensitive to perturbations of NF-M than NF-L or NF-H. However, interpretation of studies with the transgenic human protein were complicated by low expression levels in most regions including spinal cord (less than 5% of endogenous mouse NF-M) and the fact that not all mouse neurons that express NFs expressed the human NF-M protein (Lee et al., 1992). Recently we created transgenic mice that express the human NF-M protein from a bacterial artificial chromosome (BAC). These new transgenic mice express the human NF-M at about the level of the murine protein and express it broadly in most if not all neurons that express murine NF-M. Animals harboring the human NF-M BAC transgene developed a progressive hind limb paralysis. Here we describe the pathological and biochemical basis for this syndrome including findings suggesting that the human NF-M protein is toxic to mouse motor neurons.

Materials and methods Generation of transgenic mice A 143-kb human BAC clone (72M22, Sequence Accession No. AF106564) harboring the entire human NF-M gene was identified from the databases of the National Center for Biotechnology Information (NCBI) and the European Molecular Biology Organization (EMBO) using the human NF-M cDNA sequences as queries. BAC DNA for microinjection was prepared as described in detail elsewhere (Gama Sosa et al., 2002). Transgenic mice were produced by pronuclear injection using C57B1/6J ⫻ DBA/2J F1 (B6D2) hybrids as a source of fertilized eggs. Transgenic founders were identified by Southern blotting of

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genomic DNA from tail clip biopsies and subsequently by PCR. Using the primer pairs 5⬘-CTGGTGGCAGATGCCAAGGTG-3⬘ and 5⬘-TCTTCCTTGGGAGCTTCCTTGACT-3⬘ from human NF-M exon 3 (Sequence Accession No. Y00067), a 345-bp fragment was amplified in animals carrying the human transgene. As an internal control for DNA amplification a 148-bp fragment from exon 1 of the mouse myelin basic protein gene (Accession No. L00398) was coamplified using primers 5⬘-GGATGTGATGGCATCACAGAAGAGA-3⬘ and 5⬘-TGTCACCGCTAAAGAAGCGCCGAT-3⬘. All studies were performed on hemizygous transgenic mice with nontransgenic littermates used as controls. Western blotting Brains from control and transgenic animals were homogenized in 0.1 M NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM sodium phosphate, pH 6.5, supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN). The homogenates were centrifuged at 15,000g for 1 h at 20°C. After centrifugation, the supernatants were saved and the pellets were resuspended in homogenization buffer. Protein concentrations were determined in both pellet and supernatant fractions using the Bradford reagent (Bio-Rad, Richmond, CA) according to the manufacturer’s instructions. Protein samples (10 ␮g) in Laemli buffer were heat-denatured and electrophoresed through 5–12% SDS–PAGE gels and transferred onto PVDF membranes (New England Nuclear/Dupont, Boston MA). After overnight blocking with 10% newborn bovine serum/0.05% Tween-20 in Tris-buffered saline (TBS), the membranes were incubated overnight at room temperature with the primary antibody (see below) diluted in the blocking buffer described above. After extensive washes, the membranes were incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (1:200; KPL, Gaithersburg, MD) and visualized by enhanced chemoluminescence (Pierce, Rockford, IL). The following antibodies were used: rat anti-human NF-M monoclonal antibody HO-14 (1:100; gift from Dr. Virginia Lee, University of Pennsylvania) which recognizes a phosphorylated epitope found in human but not mouse NF-M (Lee et al., 1992), mouse monoclonal antibody RMO108 (1:200, Dr. Virginia Lee) which recognizes a phosphorylated epitope found in mouse but not in human NF-M (Lee et al., 1992), rabbit anti-NF-L (1:2000, Chemicon, Temecula, CA and Virginia Lee, University of Pennsylvania), mouse anti-peripherin monoclonal 1527 (1:200, Chemicon), rabbit antiNF-H extreme C-terminus (1:2000, Chemicon), mouse monoclonals anti-actin clone AC40 (1:3000, Sigma, St. Louis, MO), and mouse anti-NF-M clone NN18 (1:500, Sigma).

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Immunofluorescence staining of tissue sections Control and mutant mice were perfused transcardially with 4% phosphate-buffered paraformaldehyde. Brains were postfixed overnight in paraformaldehyde and stored in phosphate-buffered saline (PBS) at 4°C; 50-␮m-thick sections of brain and spinal cord were cut with a Vibratome (St. Louis, MO). Free floating sections were blocked for 1 h in 5% normal goat serum/0.3% Triton X-100 in TBS and incubated overnight at room temperature with the primary antibody diluted in blocking buffer. After extensive washing with PBS, sections were incubated for 1 h with the appropriate secondary antibody conjugated to Alexa-Fluor488 or Alexa-Fluor562 (Molecular Probes, Eugene, OR) diluted 1:200 in blocking buffer. Sections were mounted in glycerol with 100 mg/ml DABCO (1,4 diazabicyclo-(2,2,2)-octane) in PBS and observed on a Zeiss Axiophot fluorescence microscope. In addition to the antibodies described above, the monoclonal antibodies SMI-31 (1:200) and SMI-32 (1: 200; Sternberger Labs, Luterville, MD) as well as a rabbit anti-NF-M antibody (1:100; Chemicon) were used.

Fig. 1. Level of expression of transgene-derived human NF-M protein and hind limb weakness in 2-month-old human NF-M BAC transgenic mice. (A) Western blotting was performed on whole brain or spinal cord extracts from 2-month-old nontransgenic (⫹/⫹) or hemizygous human NF-M transgenic (Tg/⫹) animals; 10 ␮g of protein per lane was separated on a 7.5% SDS–polyacrylamide gel. Blots were probed with an antibody that recognizes both human and mouse NF-Ms. Total NF-M is elevated by about fourfold in brain and twofold in spinal cord in the transgenic animals. (B) A 2-month-old NF-M BAC transgenic mouse with a hind limb paralysis is shown. Note the abnormal posture and splaying of the hind limbs.

formed using the program StatView 5.0 (SAS Institute, Cary, NC).

Electron microscopy Tissues were processed for electron microscopy by standard methods described elsewhere (Elder et al., 1998a; Friedrich and Mugnaini, 1981). Tissues were fixed by vascular perfusion with 2% formaldehyde (from paraformaldehyde), 1% glutaraldehyde and 0.12 M sodium phosphate buffer, pH 7.4. Samples were postfixed in buffered osmium tetroxide, embedded in Epon, and examined using a JEOL 100CX electron microscope (Akashima, Japan). NFs were counted in cross-sectional images of axons photographed at a magnification of 20,000 and enlarged an additional 2.5-fold during printing. NF densities were determined as previously described (Elder et al., 1998a) by applying a template of hexagons (each equivalent to an actual area of 0.10 ␮m2) to prints of randomly chosen axons and the number of NFs that fell within alternate hexagons was counted. No more than 25 hexagons were counted in any individual axon and an average NF density was determined from the number of NFs per hexagon. Measurement of axonal diameters Axonal diameters were measured on 1-␮m-thick transverse sections of toluidine blue-stained L5 ventral root as previously described (Elder et al., 1998a). Optimal brightness and gray scale values of digital images were adjusted to provide the sharpest discrimination of the myelin/axon border and all myelinated axons were traced. Axons were assumed to be circular for purposes of diameter calculations. Statistical analysis was per-

Results Generation of transgenic mice expressing the human NF-M protein We generated transgenic mice with a BAC clone (72M22) harboring the human NF-M gene. From one of the three founder animals a line was established that had stable expression of the human transgenic protein. Western blotting on 2-month-old animals from this line with a polyclonal antibody that recognizes both the human and mouse NF-M proteins (Fig. 1A) showed that total NF-M was increased by approximately fourfold in transgenic brain and twofold in transgenic spinal cord. Western blotting with a monoclonal antibody (HO14) that recognizes human but not mouse NF-M as expected showed expression of the human protein in brain and spinal cord of transgenic animals but not in nontransgenic littermates (Fig. 6D). In addition fixed tissue sections double-stained with HO14 and the mouse NF-M specific antibody RMO108 showed identical staining patterns, confirming the widespread and neuron-specific expression pattern of the human NF-M transgene (data not shown). Motor neuron disease develops in human NF-M BAC transgenic mice Previous studies have shown that overexpressing even high levels of mouse NF-M in transgenic mice causes NF accumulations in anterior horn cells but no clinically evident motor weakness (Wong et al., 1995). The human NF-M

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BAC transgenic mice generated here also showed no obvious abnormalities at birth or in the neonatal period. However, beginning as early as 2 months of age, human NF-M BAC transgenic animals began to develop an incoordination of the hind limbs that was followed within weeks to months by a gross hind limb paralysis (Fig. 1B). The most severely affected animals became incapacitated and had to be sacrificed by 4 – 6 months of age while in the least affected animals the clinical severity was mild enough to be compatible with a nearly normal life span. Toludine blue-stained sections of transgenic brain showed no abnormalities. Rather the most prominent findings in the CNS were in the ventral horn motor neurons of the transgenic spinal cord. Many motor neurons in affected animals showed markedly enlarged perikarya and chromatolytic features (Figs. 2A and B) that were most prominent around the periphery of the cell body with the remaining Nissl stain concentrated around the nucleus. Mixed among affected neurons were normal-appearing motor neurons (Fig. 2A), indicating that the process did not affect all motor neurons simultaneously. In addition smaller interneurons in the ventral horn did not show these changes. When examined by electron microscopy the most striking feature in the perikaryon of affected neurons was a massive accumulation of 10-nm filaments around the periphery of the cell body (Figs. 2C and D) which corresponds to the chromatolytic region observed by light microscopy. Here densely packed filaments were interspersed at times with mitochondria and other cellular organelles. However, most organelles became concentrated in the perinuclear region. Proximal dendrites also appeared to contain an abnormal density of NFs, although proximal axonal segments remained fairly normal in appearance. All three mouse neurofilament subunits are present in neurofilamentous inclusions and phosphorylated mouse but not phosphorylated human NF-M accumulates in the cell body of affected neurons By immunostaining sections of spinal cord with a variety of anti-NF antibodies it was possible to determine the composition of the perikaryal accumulations (Fig. 3). For example staining of nontransgenic specimens with an antibody that recognizes both human and mouse NF-M (Figs. 3C and D) gave light staining of cell bodies and more prominent staining in dendrites and adjacent white matter axons (Fig. 3C). However, in ventral horn motor neurons of transgenic animals staining (Fig. 3D) was abnormal in both the relative intensity of cell body compared to axonal staining and the less extensive staining of dendrites. In particular, staining was apparent along the periphery of the cell body corresponding to the area of filament accumulation seen by electron microscopy. We further determined if both mouse and human NF-M proteins were accumulating with mouse- and human-specific antibodies. HO14 is an antibody that recognizes phos-

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phorylation-dependent epitopes present on the human but not the mouse NF-M (Lee et al., 1992). These epitopes are normally present on NFs found in axons but not on NFs in cell bodies (Vickers et al., 1994). On transgenic spinal cord HO14 stained axons in spinal cord white matter and many fine processes in the ventral horn but did not stain motor neuron perikarya (Fig. 3K), indicating that at least the phosphorylated form of the human NF-M was not accumulating in cell bodies to detectable levels. RMO108 is an antibody that recognizes phosphorylated forms of mouse but not human NF-M (Lee et al., 1992). Staining of nontransgenic spinal cord with this antibody produced light perikaryal staining in ventral horn motor neurons (Fig. 3I) with stronger axonal staining. However, staining of transgenic cord (Fig. 3J) revealed more prominent perikaryal and dendritic staining than in controls, indicating that phosphorylated forms of mouse NF-M were abnormally accumulating in the cell body. By immunostaining with antibodies to NF-L and NF-H it became clear that these NFs were also accumulating. On nontransgenic sections antibodies to both NF-L and NF-H showed light staining of cell bodies and more prominent staining of axons and dendrites (Figs. 3A and E) and indeed both antibodies showed prominent staining of many large dendrites of ventral horn motor neurons in normal spinal cord. By contrast in transgenic animals, staining of motor neuron cell bodies was more intense compared to axonal and dendritic staining with both antibodies and many morphologically abnormal cells were stained (Figs. 3B and F). Staining in transgenic specimens was most obvious around the periphery of the cell body and particularly the NF-H antibody revealed few stained dendrites. Staining with SMI-32 an antibody that recognizes nonphosphorylated forms of mouse NF-H as well as nonphosphorylated human NF-M (Lee et al., 1988) also revealed abnormal NF accumulation around the periphery of the cell body in transgenic tissue (Figs. 3G and H). In addition staining with an antibody SMI-31 that recognizes phosphorylated forms of mouse and human NF-M and NF-H (Lee et al., 1988) showed perikaryal staining of cells in transgenic but not nontransgenic tissue (data not shown). Due to the specificity of the SMI-31 and -32 antibodies it is not possible to determine if these antibodies are seeing proteins of human or mouse origin in the perikarya of affected motor neurons. However, these data together with the HO14 and RMO108 results described above suggest that the perikaryal epitopes recognized by the SMI-31 antibody are likely to be predominantly of mouse origin. Collectively these studies show that all three mouse NFs are contained within the NF inclusions found within affected motor neurons and that a phosphorylated form of mouse NF-M that is not normally found in cell bodies is also accumulating. It also seems likely that the human NF-M contributes to the abnormal inclusions but most likely in a predominately nonphosphorylated form. How-

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Fig. 2. Accumulation of neurofilaments in ventral horn motor neurons in human NF-M BAC transgenic mice. (A, B) Toluidine blue-stained cross sections of the ventral horns of the L2 lumbar spinal cord are shown from a 2-month-old NF-M BAC transgenic animal (Tg/⫹) with hind limb weakness (A) and a nontransgenic (⫹/⫹) littermate control (B). Note the enlarged motor neurons in the transgenic with concentration of the Nissl stain in the perinuclear region of the affected neurons such as the one indicated by the arrowhead. Examples of normal appearing cells in both the transgenic and the nontransgenic specimens are indicated by arrows. (C, D) Electron micrographs are shown of an abnormal motor neuron from a transgenic animal. In the cell body of the neuron in (C), organelles are concentrated toward the nucleus (N) and large numbers of 10-nm filaments are present in the area marked with an asterisk. The area marked with the asterisk in (C) which corresponds to the clear regions lacking Nissl stain in the cells in (A) is shown in higher power in (D). Scale bars: (A, B) 50 ␮m; (C, D) 8 ␮m in (C) and 1 ␮m in (D).

ever, since we have no antibody that can distinguish nonphosphorylated forms of the mouse and human NF-Ms, this prediction cannot be experimentally verified. ␣-internexin and peripherin are also expressed in anterior horn cells and accumulation of peripherin has been associated with experimental motor neuron disease (Beaulieu et al., 1999, 2000). Immunostaining for ␣-internexin revealed

widespread axonal staining in both transgenic and nontransgenic spinal cord but did not reveal any staining of cell bodies in either wild-type or affected transgenic animals (data not shown). With peripherin, cell bodies stained in both control and transgenic ventral horn motor neurons, but there was no discernable change in the staining intensity or pattern in the transgenic animals (data not shown).

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Fig. 3. Abnormal accumulation of neurofilament proteins in ventral horn motor neurons of human NF-M BAC transgenic mice. Immunofluorescence staining of lumbar spinal cord from 2-month-old nontransgenic (A, C, E, G, I) or clinically affected human NF-M BAC transgenic mice (B, D, F, H, J, K) is shown. Sections were stained with antibodies that recognize mouse NF-L (A, B), mouse and human NF-M (C, D), mouse NF-H (E, F), SMI-32 (G, H), an antibody that recognizes nonphosphorylated forms of mouse NF-H as well as nonphosphorylated human NF-M, RMO108 (I, J), an antibody that recognizes phosphorylated mouse but not human NF-M, and HO14 (K), an antibody that recognizes phosphorylated human but not mouse NF-M. Normal cells are indicated by arrows (A, C, E, I) and abnormal cells by arrowheads (B, D, F, J, K). In the transgenic specimens note the frequently irregular shape of the cells as well as the frequent accumulation of staining along the cell membrane such as in the cells indicated by the arrowheads in (B) and (E) which correspond to the areas containing 10-nm filaments shown in Fig. 2. Also, note the generally lesser staining of processes in the transgenic specimens especially with the NF-H antibody (compare (E) and (F)). RMO108 produces light staining of nontransgenic cell bodies (see cell indicated by the arrow in (I)) but intense staining of affected cell bodies in the transgenic section (arrowhead in (J)) By contrast, these cells do not stain with the human NF-M-specific antibody HO14 (arrowhead in (K)), indicating the preferential accumulation of phosphorylated mouse NF-M in the transgenic ventral horn cells. Scale bar: 50 ␮m.

Some motor axons are lost and remaining motor axons contain increased numbers of neurofilaments in human NF-M BAC transgenic animals If ventral horn motor neurons are being lost, motor roots should be depleted of axons. We determined the status of motor axons in human NF-M BAC transgenic mice by examining the lumbar ventral roots of affected NF-M BAC transgenic animals and nontransgenic controls (Fig. 4). The most striking feature in the ventral roots of NF-M BAC transgenic animals was a marked loss of myelinated axons. Indeed roots in affected animals contained less than half the normal number of myelinated axons (see Fig. 5A), falling from over 800 in control to less than 400 in the human NF-M BAC transgenic animals (P ⫽ 0.0004, unpaired t test). Interestingly, despite the massive axonal loss, the remaining myelinated fibers in the transgenic animals remain fairly normal in appearance. In addition only rarely were degenerating axons observed and there was no macrophage infiltration. An unusual feature was that in the transgenic roots the remaining axons appeared to assume a relatively

homogenous size distribution, unlike the broad distribution of axonal calibers seen in normal roots (Figs. 4C and D). Indeed the average diameter of the remaining axons in the NF-M BAC transgenic nerves (5.2 ⫾ 1.5 ␮m, SD) was relatively similar to that of the nontransgenic control (5.0 ⫾ 2.0 ␮m). Yet the frequency distribution of axons in the ventral roots (Fig. 5B) showed fewer large and especially fewer small myelinated axons, confirming that a relative homogenization of axonal diameters had occurred in the transgenic animals with the majority of axons falling in the 4- to 7-␮m range. Indeed the percentage of small myelinated axons less than 3 ␮m in diameter fell from 22.8% in the nontransgenic animals to only 9.6% in the NF-M BAC transgenic mice. By contrast, controls exhibited the broader bimodal distribution typically seen in normal nerves. Ultrastructurally the most striking feature in the remaining transgenic axons was an apparent increase in the number of NFs (Figs. 4E and F). Indeed measurement of NF densities (Fig. 5C) showed that NF density increased from 137/␮m2 in control axons to 186/␮m2 in the transgenic animals (P ⬍ 0.0001). In contrast to the lumbar motor roots, the lumbar sensory roots appeared normal in the NF-M

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Fig. 4. Light and electron microscope views of lumbar ventral roots from human NF-M BAC transgenic and control animals. Examples of L5 ventral roots from 2-month-old nontransgenic (A) and a clinically affected transgenic animal (B) are shown. Higher power views of myelinated axons from the roots in (A) and (B) are shown in (C) and (D). Note the relative uniformity of axonal diameter in the transgenic compared to the nontransgenic. Electron micrographs of myelinated axons from nontransgenic (E) and transgenic (F) animals show an increased density of NFs in the axons remaining in the human NF-M BAC transgenic animals. Scale bars: (C, D) 10 ␮m; (E, F) 0.25 ␮m.

transgenic animals (data not shown). Thus the lumbar sensory neurons appear to be less sensitive to perturbations caused by the human NF-M transgenic protein. Mouse NF-M is less phosphorylated and peripherin is driven out of filaments in NF-M BAC transgenic animals We utilized immunoblotting to assess whether the presence of the human NF-M altered the biochemical composi-

tion of filaments. For this purpose protein extracts from brain and spinal cord were separated into Triton X-soluble and -insoluble fractions and immunoblots were performed with a variety of anti-NF antibodies (Fig. 6). Fig. 6B shows immunoblotting with an antibody that recognizes both human and mouse NF-Ms. This antibody detects multiple species of NF-M in the transgenic sample. The slower migrating species in the transgenic sample most likely represent the human NF-M, which has a higher molecular weight than mouse NF-M due to its more extensive phosphorylation (Lee et al., 1992; Tu et al., 1995). Phosphorylation of human NF-M in mouse neurons like the native human NF-M has been previously observed in transgenic mice (Lee et al., 1992; Tu et al., 1995). The faster migrating species likely represent mouse NF-M, although it remains possible that some immunoreactivity might result from lesser phosphorylated forms of the human NF-M. From Western blotting with this antibody it was clear that total NF-M was increased in the Triton X-insoluble fractions in the transgenic animals. Only with long exposures of the blots were trace amounts of NF-M observed in the Triton X-soluble fractions, indicating that nearly all the stably accumulating NF-M protein (both human and mouse) was being incorporated into filaments. Immunoblotting with HO14 (Fig. 6D), an antibody that recognizes phosphorylated human but not mouse NF-M, showed accumulation of the human protein in the Triton X-insoluble fractions in the transgenic extracts, supporting the conclusion that it is incorporated into filaments with the mouse NFs. We next determined what effect the presence of the transgenic human protein had on the incorporation of mouse NF-M into filaments by immunoblotting with antibody RM0108 (Fig. 6E), which recognizes phosphorylated forms of mouse but not human NF-M (Lee et al., 1992). Blotting with this antibody showed that, as with the transgenic human protein, all the detectable mouse NF-M was incorporated into the pellet fraction. In addition the total amount of mouse NF-M in the pellet did not appear to be significantly altered between the transgenic and the nontransgenic animals. However, the pattern of phosphorylation of the mouse NF-M was altered, with lesser amounts of a slowly migrating band representing the most phosphorylated forms of the mouse NF-M and the appearance of a new faster migrating band that was not seen in nontransgenic brain or spinal cord. The appearance of this faster migrating band argues that the human transgene is likely competing with the endogenous mouse NF-M for phosphorylation and is shifting the endogenous mouse NF-M to a lesser phosphorylated state. This finding also likely explains why the major presumed mouse NF-M band in Fig. 6B is migrating at a faster rate in the transgenic specimens. The lack of any significant appearance of either mouse or human NF-Ms in the soluble fraction with either the human or mouse specific antibodies again argues that essentially all mouse and human NF-M that is stably accumulating is incorporated into filaments.

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Fig. 5. Loss of myelinated axons and increased density of neurofilaments in remaining axons in the lumbar ventral roots of NF-M BAC transgenic mice. (A) Myelinated axons were counted in the L5 ventral roots of clinically affected 2-month-old NF-M BAC transgenic (Tg/⫹, n ⫽ 4 roots from three animals) or unaffected littermate controls (⫹/⫹, n ⫽ 3 roots from two animals). The NF-M BAC transgenic had significantly fewer myelinated axons than the controls (P ⫽ 0.004, unpaired t test). (B) Diameters of all myelinated axons were measured in the L5 ventral roots from NF-M transgenic or control animals (n ⫽ 2 roots per genotype). Result are presented as the percentage distribution of all measured axons (n ⫽ 1588 nontransgenic and 715 transgenic). Note the narrowed distribution of myelinated fibers in the transgenic. (C) NF densities were determined by applying a template of hexagons over electron micrographs of myelinated fibers from the L5 ventral roots and counting the number of NFs in randomly placed hexagons (see Materials and Methods). At least 300 hexagons (n ⫽ 311 transgenic and 313 nontransgenic), each equivalent to an area of 0.10 ␮m2, were counted for each group and a frequency distribution plot was generated. Density was increased from an average of 13.7 ⫾ 5.8 (SD) in the nontransgenic to 18.6 ⫾ 7.3 in the NF-M transgenic axons (P ⬍ 0.0001).

Along with increased total NF-M in the Triton X-insoluble fractions, we also observed increased amounts of mouse NF-L and NF-H (Figs. 6A and C), arguing that overexpression of the human NF-M is also driving more NF-L and NF-H into assembled filaments. These findings are consistent with the increased accumulation of filaments in the cell bodies of transgenic animals and the increased density of filaments in transgenic axons. Interestingly changes were seen in both spinal cord and brain despite the fact that we have only rarely observed accumulations of NFs in even large brainstem motor neurons by immunocytochemistry (data not shown). Peripherin has also been associated with filamentous inclusions in human and experimental models of motor neuron disease (Beaulieu et al., 1999, 2000). Interestingly in the human NF-M BAC transgenic animals, peripherin was decreased in the Triton X-insoluble fraction (Fig. 6F) arguing that the human NF-M is driving peripherin out of filaments.

No relief of symptoms when the human NF-M transgene is expressed on the murine NF-M heterozygous null background Elevation of total NF-M (i.e., mouse plus human) would be expected to alter the basic stoichiometry of NFs in the cell and might lead to NF accumulations. Supporting this notion, motor neuron disease caused by overexpression of human NF-H can be rescued by overexpressing it in combination with a human NF-L transgene that restores the correct stoichiometry between NF-L and NF-H subunits (Meier et al., 1999). If stoichiometry is key to the disease described here, then removing some or all of the endogenous mouse NF-M might lessen the pathology. We therefore bred the human NF-M BAC transgenic mice with mice harboring a null mutation in the mouse NF-M gene (Elder et al., 1998a). Since in transgenic spinal cord total NF-M is elevated by approximately twofold (Fig. 1), complete transfer of the human transgene onto the mouse

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null mutant (C57B1/6) make it difficult to directly compare the speed of development of paralysis on the mouse NF-M ⫹/⫹ vs ⫹/⫺ background. Nevertheless, it is clear that removing half of the mouse NF-M offers no significant protection against the development of disease associated with the human NF-M transgene and may even accelerate the rate of development of pathology. Discussion

Fig. 6. Increased incorporation of NFs into Triton X-insoluble fractions in human NF-M BAC transgenic mice. Brain and spinal cord extracts from two month old animals were made in Triton X-100 and the Triton X-insoluble pellets and supernatants were collected. Equal amounts (10 ␮g) of protein per lane were electrophoresed through SDS–PAGE gels. Western blotting was performed on total extracts (Total), and the Triton X-insoluble (Pellet) and soluble (Sup) fractions from brain or spinal cord of two-monthold non-Tg (⫹/⫹) or hemizygous human NF-M transgenic (Tg) animals. Blots were probed with an antibody that recognizes NF-L (A), human and mouse NF-M (B), NF-H (C), HO14 (D) which recognizes phosphorylated human but not mouse NF-M, RMO108 (E) which recognizes phosphorylated mouse but not human NF-M, peripherin (F), and ␤-actin (G).

NF-M null background should effectively restore normal levels of NF-M protein. The first round of breeding produced mice that contained the human NF-M transgene on the murine heterozygous null background. Interestingly all hemizygous transgenic/heterozygous (⫹/⫺) null mice from these crosses developed a hind limb paralysis within 2 months of age that was identical to that on the wild-type background. Pathology in these mice was found to be identical to that which developed typically more slowly on the ⫹/⫹ background (Fig. 7). Indeed, we have not been able to breed the hemizygous transgenic/heterozygous null mutants in order to establish the human transgene on a mouse NF-M null background. This is distinctly unlike the NF-M transgenics on the wild-type mouse background, which typically breed effectively. The differing genetic backgrounds of the human NF-M BAC transgenic (B6D2) and murine NF-M

Accumulation of NFs in the cell body and proximal axon is a core pathological feature of human motor neuron disease (Julien and Beaulieu, 2000). Experimentally overexpression of mouse NF-L (Xu et al., 1993), a mouse NF-L rod domain mutant (Lee et al., 1994), human NF-H (Cote et al., 1993), and mouse peripherin (Beaulieu et al., 1999) can cause motor neuron disease in transgenic mice. Here we describe for the first time motor neuron disease in transgenic mice expressing the human NF-M. Previous animals generated with a smaller 8.5-kb genomic human NF-M transgene (Lee et al., 1992) developed age-dependent accumulations of NFs in neocortex (Vickers et al., 1994) but never developed motor symptoms. We suspect that the lack of motor disease reflects the relatively low expression of the human NF-M protein in the spinal cord of these animals, which never exceeded more than 5% of the endogenous mouse NF-M (Lee et al., 1992). By contrast, the human NF-M BAC clone utilized here created levels of human protein likely equaling or exceeding the endogenous mouse.

Fig. 7. Ventral horn cell pathology and accumulation of neurofilaments in motor neurons in mice in which the human NF-M BAC transgene is expressed on the mouse heterozygous null background. (A, B) Examples of toludine blue-stained ventral horn motor neurons from a 6-week-old hemizygous transgenic/heterozygous NF-M null mutant (A) and heterozygous null mutant controls (B) are shown. (C, D) Sections from hemizygous transgenic/heterozygous null animals were immunostained with anti-NF-L (C) or SMI-32 (D) as in Fig. 3. Note the similar morphology of affected neurons to those in Fig. 2 and 3. Scale bar: 25 ␮m.

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The core pathological change in the human NF-M BAC transgenic mice was a massive accumulation of 10-nm filaments within the cell bodies of ventral horn motor neurons. Indeed the perikarya of motor neurons in most experimental models of motor neuron disease contain accumulations of 10-nm filaments, suggesting that filament accumulation might block cellular transport mechanisms, leading to axonal atrophy and motor neuron death. Yet overexpression of mouse NF-M (Wong et al., 1995), mouse NF-H (Marszalek et al., 1996), or a mouse NF-H LacZ transgene (Eyer and Peterson, 1994) also cause massive NF accumulations in ventral horn motor neurons without overt motor disease. Additionally mice that express a human NF-H transgene on an NF-L null background develop motor neuron disease but exhibit only nonfilamentous perikaryal inclusions (Beaulieu et al., 2000), suggesting that the composition of the accumulations may be more important than the presence or absence of filaments. One issue that must be addressed in all transgenic models in which a human protein is expressed on the background of a homologous mouse protein is whether the elevation of total protein itself (i.e., mouse plus human) may be toxic. NFs assemble in precise molar ratios and NF stoichiometry is critical in determining axonal diameter (Xu et al., 1996) and also influences the organization of NFs in dendrites (Kong et al., 1998). Thus disordered subunit ratios might be toxic to motor neurons independent of aberrant filament formation. Supporting this notion, motor neuron disease caused by overexpression of human NF-H can be rescued by expressing it in combination with a human NF-L transgene that restores the correct molar ratios between NF-L and NF-H (Meier et al., 1999). It seems unlikely, however, that human NF-M-associated motor neuron disease can be explained by stoichiometry alone, since the disease described here was in no way lessened when half the mouse NF-M was removed by breeding the human NF-M transgene onto the mouse heterozygous null NF-M background. Indeed it is of interest to compare our results to those of Wong et al. (1995), who overexpressed the mouse NF-M in transgenic mice. Despite an approximate doubling of mouse NF-M protein, these animals exhibited no overt phenotype up to 24 months of age despite NF swellings in perikarya and proximal motor axons. Seen in this context our studies are more suggestive that the human NF-M protein exerts a toxic effect on mouse motor neurons. Interestingly, similar arguments have been made concerning toxicity of the human NF-H protein (Marszalek et al., 1996). Specifically, while expressing modest levels of human NF-H in transgenic mice results in motor neuron disease (Cote et al., 1993), expressing mouse NF-H at similar or higher levels does not result in any overt phenotype despite marked perikaryal NF accumulations (Marszalek et al., 1996). Collectively, these results suggest that the two largest human NF proteins may be toxic to mouse motor neurons. Why the human protein might be toxic is unclear, although the biochemical studies described here indicate that

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the mouse and human proteins are not processed equivalently within the mouse neuron. Both NF-M and NF-H have long sequence extensions on the COOH terminal side of their rod domains (Lees et al., 1988; Levy et al., 1987; Myers et al., 1987; Shneidman et al., 1988). These additional regions project from the NF backbone and likely determine the surface properties of NFs (Hirokawa et al., 1984; Mulligan et al., 1991). Structurally human NF-M (Myers et al., 1987) differs from rodent NF-Ms (Levy et al., 1987; Napolitano et al., 1987) in having within its COOH terminal region a sequence Lys-Ser-Pro-Val-Pro-Lys-SerPro-Val-Glu-Glu-Lys-Gly that is repeated serially six times yielding a 78-amino acid unit in which Lys-Ser-Pro-Val (KSPV) occurs 12 times. These KSPV sequences are phosphorylated in the human NF-M protein (Lee et al., 1988). By contrast only a single copy of a similar sequence is found in rat (Napolitano et al., 1987) or mouse (Levy et al., 1987) NF-Ms yielding two KSPVs in rat and one KSPV and one KSPM in mouse. Mouse NFs and human NF-M are capable of copolymerization both in vitro, after transfection into cultured cells (Carter et al., 1998), and in vivo in transgenic animals (Lee et al., 1992). The studies described here also show that all NF-M protein whether mouse or human partitions into the Triton X-insoluble fraction. Indeed, levels of all the endogenous mouse NF proteins were increased in the human NF-M BAC transgenic animals, arguing against any notion that the human may more effectively compete for assembly into filaments than the endogenous mouse. However, the presence of the additional KSPV sites on the human NF-M may account for the hypophosphorylation of the mouse NF-M seen here if they make the human NF-M a more efficient substrate for phosphorylation. More efficient phosphorylation might also explain the more efficient transport of the human protein, as evidenced by the accumulation of phosphorylated mouse but not phosphorylated human NF-M in the cell bodies of affected motor neurons. Why hypophosphorylation of mouse NF-M should prove detrimental is unclear. Mice lacking NF-M do not exhibit motor neuron disease (Elder et al., 1998a). Mice lacking both NF-M and NF-H do with aging develop motor system disease but the pathological basis of this syndrome is a progressive depletion of NFs from nerve roots rather than accumulations of NFs in ventral motor neurons (Elder et al., 1999). Rather collectively these studies more likely suggest a toxic role for the human NF-M protein, perhaps leading to a deleterious accumulation of partially phosphorylated mouse NF-M in the cell body. Interestingly surviving motor axons in NF-M BAC transgenic animals contain an increased density of NFs. An increased density of NFs in CNS axons of human NF-M transgenic mice has also been observed previously (Tu et al., 1995). This increase could reflect either an increase of total NF-M or be a selective effect of the human NF-M protein. Arguing against it being simply an effect of total NF-M expression is the observation that a similar overex-

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pression of mouse NF-M in transgenic animals leads to no difference in NF packing density in ventral root axons (Wong et al., 1995). Thus the presence of the human NF-M likely alters NF assembly properties in the mouse, leading to an increased density of axonal NFs, at least in those neurons that can tolerate the chronic overexpression of the human protein. Further supporting the notion that assembly properties are altered, the human NF-M also drives peripherin out of filaments in the transgenic animals. Peripherin overexpression has been associated with motor neuron disease in transgenic animals (Beaulieu et al., 1999). Any direct pathogenic role for peripherin’s exclusion here remains unclear. However, the preferential loss of smaller diameter fibers in the NF-M BAC transgenic animals is interesting, given peripherin’s concentration in smaller caliber axons (Brody et al., 1989), and suggests that the human NF-M may be selectively killing neurons with the highest endogenous levels of peripherin. Anterior horn cells seem especially vulnerable to perturbations of intermediate filament proteins. The basis for this selectivity remains unknown. The present studies add human NF-M to the list of intermediate filament proteins that can produce motor neuron disease in the mouse. However, they also raise a cautionary note concerning the use of transgenic mice to model the normal biological functions of human cytoskeletal proteins such as NF-M or NF-H. If indeed other cytoskeletal proteins behave in a similar fashion, it may prove difficult in other settings as well to distinguish toxicity of a human protein from the effects of altered level of expression. It may also mean that a truly functional study of the human proteins in the mouse would be possible only by establishing all three human NF proteins in the mouse as transgenes in the absence of the mouse NFs. The latter issue may be highly relevant to establishing the mouse as a model system for human ALS.

Acknowledgments This work was supported by National Institutes of Health Grant P50 AG05138. We thank Ms. Valerie Williams and the Mount Sinai Microscopy Shared Research Facility for assistance with electron microscopy and Ms. Gissel Perez for technical assistance.

References Beaulieu, J.M., Jacomy, H., Julien, J.P., 2000. Formation of intermediate filament protein aggregates with disparate effects in two transgenic mouse models lacking the neurofilament light subunit. J. Neurosci. 20, 5321–5328. Beaulieu, J.M., Nguyen, M.D., Julien, J.P., 1999. Late onset death of motor neurons in mice overexpressing wild-type peripherin. J. Cell Biol. 147, 531–544.

Brody, B.A., Ley, C.A., Parysek, L.M., 1989. Selective distribution of the 57-kDa neural intermediate filament protein in the rat CNS. J. Neurosci. 9, 2391–2404. Carter, J., Gragerov, A., Konvicka, K., Elder, G., Weinstein, H., Lazzarini, R.A., 1998. Neurofilament assembly: divergent characteristics of human and rodent NF-L subunits. J. Biol. Chem. 273, 5101–5108. Cote, F., Collard, J.-F., Julien, J.-P., 1993. Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis. Cell 73, 35– 46. Elder, G.A., Friedrich, V.L., Bosco, P., Kang, C., Gourov, A., Tu, P.-H., Lee, V.M.-Y., Lazzarini, R.A., 1998a. Absence of the mid-sized neurofilament subunit decreases axonal diameter, levels of light neurofilament (NF-L) and neurofilament content. J. Cell Biol. 141, 727–739. Elder, G.A., Friedrich, V.L., Kang, C., Bosco, P., Gourov, A., Tu, P.-H., B, Z., Lee, V.M.-Y., Lazzarini, R.A., 1998b. Requirement of heavy neurofilament subunit in the development of axons with large calibers. J. Cell Biol. 143, 195–205. Elder, G.A., Friedrich Jr., V.L., Margita, A., Lazzarini, R.A., 1999. Agerelated atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit. J. Cell Biol. 146, 181–192. Eyer, J., Peterson, A., 1994. Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-betagalactosidase fusion protein. Neuron 12, 389 – 405. Friedrich, V.L.J., Mugnaini, E. 1981. Preparation of neural tissues for electron microscopy, in: A Handbook of Neuroanatomical Tract Tracing Techniques, Plenum Press, New York, pp. 377– 406. Gama Sosa, M.A., DeGasperi, R., Gonzalez, E.A., Kelley, K., Lazzarini, R., Elder, G.A., 2002. BAC and PAC DNA for the generation of transgenic animals. Biotechniques 33, 51–53. Hirokawa, N., Glicksman, M.A., Willard, M.B., 1984. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J. Cell Biol. 98, 1523–1536. Julien, J.P., Beaulieu, J.M., 2000. Cytoskeletal abnormalities in amyotrophic lateral sclerosis: beneficial or detrimental effects? J. Neurol. Sci. 180, 7–14. Julien, J.-P., Meyer, D., Flavell, D., Hurst, J., Grosveld, F., 1986. Cloning and developmental expression of the murine neurofilament gene family. Mol. Brain Res. 1, 243–250. Kong, J., Tung, V.W., Aghajanian, J., Xu, Z., 1998. Antagonistic roles of neurofilament subunits NF-H and NF-M against NF-L in shaping dendritic arborization in spinal motor neurons. J. Cell Biol. 140, 1167– 1176. Lee, M.K., Cleveland, D.W., 1996. Neuronal intermediate filaments, in: Cowan, W.M., Shotter, E.M., Stevens, C.F., Thompson, R.F. (Eds.), Annual Review of Neuroscience, Annual Reviews, Inc., Palo Alto, CA, pp. 187–217. Lee, M.K., Marszalek, J.R., Cleveland, D.W., 1994. A mutant neurofilament subunit causes massive, selective motor neuron death: implications for the pathogenesis of human motor neuron disease. Neuron 13, 975–988. Lee, V.M.-Y., Elder, G.A., Chen, L.-C., Liang, Z., Snyder, S.E., Friedrich, V.L., Lazzarini, R.A., 1992. Expression of human mid-sized neurofilament subunit in transgenic mice. Mol. Brain Res. 15, 76 – 84. Lee, V.M.-Y., Otvos Jr., L., Carden, M., Hollosi, M., Dietzschold, B., Lazzarini, R.A., 1988. Identification of the major multiphosphorylation site in mammalian neurofilaments. Proc. Natl. Acad. Sci. USA 85, 1998 –2002. Lees, J.F., Shneidman, P.S., Skuntz, S.F., Carden, M.J., Lazzarini, R.A., 1988. The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it. EMBO J. 7, 1947–1955. Levy, E., Liem, R.K.H., D’Eustachio, P., Cowan, N.J., 1987. Structure and evolutionary origin of the gene encoding mouse NF-M, the middlemolecular-mass neurofilament protein. Eur. J. Biochem. 166, 71–77. Marszalek, J.R., Williamson, T.L., Lee, M.K., Xu, Z., Hoffman, P.N., Becher, M.W., Crawford, T.O., Cleveland, D.W., 1996. Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport. J. Cell Biol. 135, 711–724.

M.A. Gama Sosa et al. / Experimental Neurology 184 (2003) 408 – 419 Meier, J., Couillard-Despres, S., Jacomy, H., Gravel, C., Julien, J.P., 1999. Extra neurofilament NF-L subunits rescue motor neuron disease caused by overexpression of the human NF-H gene in mice. J. Neuropathol. Exp. Neurol. 58, 1099 –1110. Mulligan, L., Balin, B.J., Lee, V.M.-Y., Ip, W.P., 1991. Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms. J. Struct. Biol. 106, 145–160. Myers, M.W., Lazzarini, R.A., Lee, V.M.-Y., Schlaepfer, W.W., Nelson, D.L., 1987. The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family. EMBO J. 6, 1617–1626. Napolitano, E.W., Chin, S.S.M., Colman, D.R., Liem, R.K.H., 1987. Complete amino acid sequence and in vitro expression of rat NF-M, the middle molecular weight neurofilament protein. J. Neurosci. 7, 2590 – 2599. Rao, M.V., Houseweart, M.K., Williamson, T.L., Crawford, T.O., Folmer, J., Cleveland, D.W., 1998. Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament Heavy subunit (NF-H) or its phosphorylation. J. Cell Biol. 143, 171–181. Shneidman, P.S., Carden, M.J., Lees, J.F., Lazzarini, R.A., 1988. The structure of the largest murine neurofilament protein (NF-H) as revealed by cDNA and genomic sequences. Mol. Brain Res. 4, 217–231. Tu, P.H., Elder, G., Lazzarini, R.A., Nelson, D., Trojanowski, J.Q., Lee, V.M., 1995. Overexpression of the human NFM subunit in transgenic

419

mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits. J. Cell Biol. 129, 1629 –1640. Vickers, J.C., Morrison, J.H., Friedrich Jr., V.L., 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. Wong, P.C., Marszalek, J., Crawford, T.O., Xu, Z., Hsieh, S.T., Griffin, J.W., Cleveland, D.W., 1995. Increasing neurofilament subunit NF-M expression reduces axonal NF-H, inhibits radial growth, and results in neurofilamentous accumulation in motor neurons. J. Cell Biol. 130, 1413–1422. Xu, Z., Cork, L.C., Griffin, J.W., Cleveland, D.W., 1993. Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease. Cell 73, 23–33. Xu, Z., Marszalek, J.R., Lee, M.K., Wong, P.C., Folmer, J., Crawford, T.O., Hsieh, S.T., Griffin, J.W., Cleveland, D.W., 1996. Subunit composition of neurofilaments specifies axonal diameter. J. Cell Biol. 133, 1061–1069. Zhu, Q., Couillard-Despres, S., Julien, J.P., 1997. Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp. Neurol. 148, 299 –316. Zhu, Q., Lindenbaum, M., Levavasseur, F., Jacomy, H., Julien, J.P., 1998. Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta,beta⬘-iminodipropionitrile. J. Cell Biol. 143, 183–193.