Motor neurons and neurofilaments in sickness and in health

Cell, Vol. 73, 1-3, April 9, 1993, Copyright

0 1993 by Cell Press

Motor Neurons and Neurofilaments in Sickness and in Health Scott T. Brady Department of Cell Biology and Neuroscience University of Texas Southwestern Medical Center Dallas, Texas 752359039

Neurofilaments were described more than 100 years ago, but they remain enigmatic. Long before a molecular identity existed, neurofilaments played a key role in creating modern neuroscience. In neuroanatomy from the time of Cajal to the present, histochemical stains have been used to visualize neurons by deposition of metals onto fibrillar structures. These discrete patterns helped establish the cellular nature of neurons and were later found to be due to 10 nm filaments abundant in neurons. The importance of neurofilaments for neuronal morphology is well documented, but their modulation in development, regeneration, and maintenance of nerves remains poorly understood. Neurofilament functions may overlap with other cytoskeletal elements, and biochemical modifications may affect their roles (Brady, 1992). Neurofilaments are neuron-specific cytoskeletal elements that are 10 nm in diameter and many microns long (Steiner-t and Roop, 1988). Initial identification of neurofilament subunits was problematic because neurofilaments are difficult to solubilize and no neurofilament ligands were available. Discovery of neurofilament subunits came by an unexpected route. Five polypeptides comprised >70% of material in the slowest axonal transport rate, known as slow component a (Hoffman and Lasek, 1975). Two are tubulins, but the other three were unknown proteins of molecular mass 200 kd, 180 kd, and 88 kd. The proposal that this triplet represented neurofilament subunits (NF-H, NF-M, and NF-L, respectively) was confirmed and extended by biochemical studies (Schlaepfer and Freeman, 1978). Both neurofilaments and microtubules move as discrete cytological structures. While radiolabel studies (Lasek et al., 1992) and direct observation of individual cytoskeletal structures (Reinsch et al., 1991) indicate that microtubules and neurofilaments move down the axon, the motor involved is unknown (Brady, 1991). Little degradation of neurofilaments occurs until they reach nerve terminals, where neurofilaments are rapidly degraded (Garner, 1988; Paggi and Lasek, 1987). Changes in axonal transport combine with differential metabolism to target and regulate the cytoskeleton. If degradative rates are constant, cytoskeletal composition in a given location is determined by the amount transported and rates of transport. Changes in expression and axonal transport rates lead to dramatic alteration of neurofilament levels during development and regeneration (Hoffman et al., 1988). When delivery by transport is constant, cytoskeletal proteins degraded slowly accumulate to higher steady-state concentrations than proteins with rapid degradation. For example, high levels of actin compared with that of neurofilaments in synaptic terminals may be ex-

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plained by low turnover of actin relative to neurofilament protein (Garner, 1988; Paggi and Lasek, 1987). Futthermore, inhibition of neurofilament proteolysis produces neurofilament rings in synaptic terminals (Roots, 1983). Differential turnover may be achieved by specific proteases or posttranslational modifications that affect degradation rates. The metabolic stability of neurofilaments suggests a cytoarchitectonic role (Lasek, 1988). Neurofilaments help determine axonal caliber by changes in neurofilament number and phosphorylation (Lasek, 1988), both of which are regulated during development (Hoffman et al., 1988). NF-M and NF-H are highly phosphorylated on sidearms formed by their carboxyl termini (Lee et al., 1988) with up to 50 phosphates per NF-H subunit. Although sidearms are often called cross-bridges, no evidence exists for cross-links between neurofilaments (Brown and Lasek, 1990; Price et al., 1988). Indeed, the charge density on phosphorylated neurofilament surfaces makes stable interactions with structures of like charge (i.e., other phosphorylated neurofilaments) improbable, and neurofilament surface charge may modulate spacing between adjacent neurofilaments (de Waegh et al., 1992). Neurofilaments are implicated in the etiology of various pathologies. Disruption of neurofilament organization is diagnostic for many neuropathologies, including motor neuron disease (Hirano, 1991), neurodegenerative diseases (Goldman and Yen, 1988), and toxin-induced neuropathies(Griffin and Watson, 1988). Therefore, the effect of overexpressing neurofilament protein in transgenic mice has been examined. Initial results were disappointing, as increased NF-L expression produced more neurofilaments but no obvious pathology (Monteiro et al., 1990). Two reports (Cot6 et al., 1993; Xu et al., 1993 [this issue of C&//j) now show that overexpression of neurofilament subunits produces a condition resembling amyotrophic lateral sclerosis (ALS). ALS is a disease of motor neurons, known as Lou Gehrig’s disease and more recently as the disease afflicting physicist Stephen Hawkings. Over months to years, ALS leads to complete paralysis and eventual death (Norris, 1992). Clinically, ALS is a progressive muscle atrophy resulting from loss of spinal and cortical motor neurons. Motor neurons giving rise to the largest myelinated axons are most severely affected, and other neuronal populations are spared (Hirano, 1991). The selectivity of ALS is striking, but little was known about its etiology. Both hereditary and sporadic forms of ALS exist, with the sporadic form representing 900/b of all cases. Xu et al. (1993) have used a mouse wild-type NF-L gene with a viral promoter to produce transgenic mice. In two founder lines, NF-L expression is doubled, and crosses between parental strains produce doubly transgenic mice expressing four times the normal NF-L levels. In these animals, large motor neuronsdevelop perikaryal neurofilament accumulations with phosphorylated NF-H. The number of degenerating axons increases, and axon neurofila-

ment densities are high. Muscles innervated by large motor neurons exhibit severe atrophy coincident with accumulation of perikaryal neurofilaments. Most doubly transgenic animals die in the third week, but surviving animals gradually recover, as transgene expression is reduced and neurofilament levels return to near normal. C&e et al. (1993) have introduced the human NF-H gene and regulatory elements into mice. Total NF-H expression levels are 3 to 4-fold normal in spinal cord and somewhat less in other regions. NF-H transgenics are less severely affected than NF-L transgenics (Xu et al., 1993) with onset of overt pathology delayed until l-4 months of age. Differences in onset and severity may reflect differences in NF-H and NF-L expression patterns. NF-H expression increases later in development than endogenous NF-L (Hoffman et al., 1988) or transgenic NF-L (Monteiro et al., 1990). Both NF-H and NF-L transgenics exhibit muscle atrophy, but many NF-H transgenic axons have reduced caliber, increased myelin thickness, and a paucity of neurofilaments. This aspect of NF-H transgenic mice is atypical of ALS and NF-L transgenic cytopathology, in which demyelination and degeneration are more frequent. Nevertheless, both NF-L and NF-H transgenics exhibit characteristics of ALS, including cytoskeletal inclusions, muscle atrophy, and loss of large motor neurons. To complicate the situation, a concurrent report has identified the lesion in a subset of familial ALS cases as a mutation in the copper/zinc superoxide dismutase (SOD7) gene (Rosen et al., 1993). Since enzyme activities have not yet been examined, mutant SOD1 enzyme may be either less active (increasing oxygen free radical levels and hypersensitivity to oxidative cell damage) or more active (increasing cell damage by elevation of H202 and hydroxyl radicals). The unusual vulnerability of motor neurons to these defects remains to be understood but may reflect specific aspects of motor neuron metabolism, such as excitotoxicity and oxidative metabolic pathways. How can such different molecular mechanisms produce similar pathologies, including loss of specific motor neurons? A trivial explanation is that both pathways result in the death of large motor neurons, but these genes are widely expressed in other neurons and even nonneuronal cells (in the case of SOD7). Yet in both SOD7 familial ALS and neurofilament transgenic mice, other cell populations are unaffected, or the changes may not result in obvious pathology. Development of motor neuron disease in transgenic mice overexpressing neurofilament subunits is plausible, because hallmarks of ALS pathology include neurofilament inclusions in affected motor neurons (Hirano, 1991) and altered neurofilament phosphorylation (Manetto et al., 1988). Neurofilament densities are often higher than normal, but axonal neurofilaments retain a parallel organization. The similar pathology produced by overproduction of different Furofilament subunits restricts plausible mechanisms. One attractive model is that increased neurofilaments interfere with other pathways such as fast axonal transport, resulting in a failure to maintain axonal struc-

tures or trophic factor supply. Consistent with this model, there is an apparent increase in mitochondrial density in transgenic mouse axons (C&e et al., 1993; Xu et al., 1993) and in a mouse model of Charcot-Marie-Tooth disease with increased neurofilament densities (de Waegh et al., 1992). However, less specific mechanisms may also be invoked. Although 3-to Cfold overexpression of transgenes may seem modest, neurofilament gene products are among the most abundant proteins of large motor neurons. Similar levels of overexpression (3- to 5fold) of rhodopsin in retinal photoreceptors lead to specific loss of photoreceptors (Olsson et al., 1992). Significant increases in production of major cell-specific gene products may constitute a severe metabolic stress, leading to specific losses in the most vulnerable cell populations. Pathology is likely to stem from overexpression of neurofilament subunits rather than increased neurofilament densities or cross-linking. First, NF-L is not implicated in formation of neurofilament sidearms, yet comparable overexpression of either NF-L or NF-H produces similar losses in motor neurons, accumulation of neurofilaments, and muscle atrophy (Cot6 et al., 1993; Xu et al., 1993). Second, cytoskeletal inclusions and increased neurofilament densities in axons may occur without creating muscle atrophy. For example, demyelination producescomparable increases in neurofilament density and reduced NF-H phosphotylation (de Waegh et al., 1992) but does not produce ALSlike pathology. Regardless, the molecular basis for loss of motor neurons in neurofilament transgenics is likely to differ from the cause of losses due to SOD1 mutations. The progression of ALS is highly variable in the severity, timing, and order in which clinical symptoms appear, so questions arise as to whether ALS is a single disease or a set of largely unrelated diseases with a common target, the motor neuron (Norris, 1992). Certainly a number of closely related pathological conditions exist. More than 70 different heritable motor neuron diseases have been described that affect both juveniles and adults, including autosomal dominant, autosomal recessive, and X-linked forms (Rowland, 1991). Only a small fraction of known hereditary motor neuron diseases have been related to specific genetic lesions, and the etiology of sporadic ALS remains unclear. However, the data clearly suggest a wide range of underlying causes. The good news is that both sets of studies have identified molecular defects that may result in ALS for defined patient populations. The bad news is that the biological mechanisms for producing motor neuron death by neurofilament overexpression and SOD7 mutations are so different that clinical interventions that help one group may be of little value to other victims of ALS. Nevertheless, these three reports have focused attention on molecular mechanisms for loss of specific cell populations and illustrate that subtle differences in regulation of gene expression for proteins considered to be “housekeeping” genes may affect survival of specific cell populations.

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