Search PubMed⌕ Search

Biomedical subjects

D W Cleveland

Publications and source records attributed to D W Cleveland.

At least 19 recordsLinked to original sources
PubMed1996

A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly.

NuMA is a nuclear protein during interphase but redistributes to the spindle poles early in mitosis. To investigate its role during spindle formation, we tested spindle assembly in frog egg extracts from which NuMA was immunodepleted. Immunodepletion revealed that NuMA forms a complex with cytoplasmic dynein and dynactin. The depleted extracts failed to assemble normal mitotic spindles, producing, instead, chromatin-associated irregular arrays of microtubules lacking characteristic spindle poles. A subdomain of the NuMA tail was shown to induce microtubule aster formation by mediating microtubule bundling. Our findings suggest that NuMA forms bifunctional complexes with cytoplasmic dynein and dynactin that can tether microtubules at the spindle poles and that are essential for mitotic spindle pole assembly and stabilization.

Amino Acid Sequence↗
PubMed1996

An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments.

Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.

Actin Cytoskeleton↗
PubMed1996

Cytoplasmic O-GlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H.

Neurofilaments, the major intermediate filaments in large myelinated neurons, are essential for specifying proper axonal caliber. Mammalian neurofilaments are obligate heteropolymers assembled from three polypeptides, neurofilament (NF)-H, NF-M, and NF-L, each of which undergoes phosphorylation at multiple sites. NF-M and NF-L are known to be modified by O-linked N-acetylglucosamine (O-GlcNAc) (Dong, D. L.-Y., Xu, Z.-S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) J. Biol. Chem. 268, 16679-16687). Here we further report that NF-H is extensively modified by O-GlcNAc at Thr53, Ser54, and Ser56 in the head domain and, somewhat surprisingly, at multiple sites within the Lys-Ser-Pro repeat motif in the tail domain, a region in assembled neurofilaments known to be nearly stoichiometrically phosphorylated on each of the approximately 50 KSP repeats. Beyond the earlier identified sites on NF-M and NF-L, O-GlcNAc sites on Thr19 and Ser34 of NF-M and Ser34 and Ser48 of NF-L are also determined here, all of which are localized in head domain sequences critical for filament assembly. The proximity of O-GlcNAc and phosphorylation sites in both head and tail domains of each subunit indicates that these modifications may influence one another and play a role in filament assembly and network formation.

Acetylglucosamine↗
PubMed1996

Motor neuron disease and model systems: aetiologies, mechanisms and therapies.

The phenotypes of many neurological diseases, including motor neuron disease (amyotrophic lateral sclerosis; ALS) and Alzheimer's disease (AD), are determined by the vulnerabilities of populations of nerve cells and the character/ evolution of cellular abnormalities. Because different cell types respond selectively to individual trophic factors, these factors may be useful in ameliorating pathology in cells that express their cognate receptors. To test therapies for ALS and AD, investigators require model systems. Although there are a variety of models of ALS, two models are particularly attractive: transgenic mice that express human superoxide dismutase 1 (SOD-1) mutations linked to familial ALS develop paralysis associated with a gain of adverse property of the mutant SOD; and axotomy of facial axons in neonatal rats, a manipulation that causes retrograde cell degeneration, which can be ameliorated by several trophic factors.

Animals↗
PubMed1996

Sequence variants in human neurofilament proteins: absence of linkage to familial amyotrophic lateral sclerosis.

Neurofilaments, assembled from NF-L (68 kd), NF-M (95 kd), and NF-H (115 kd), are the most abundant structural components in large myelinated axons, particularly those of motor neurons. Aberrant neurofilament accumulation in cell bodies and axons of motor neurons is a prominent pathological feature of several motor neuron diseases, including sporadic and familial amyotrophic lateral sclerosis (ALS). Transgenic methods have proved in mice that mutation in or increased expression of neurofilament subunits can be primary causes of motor neuron disease that mimics the neurofilamentous pathology often reported in human disease. To examine whether mutation in neurofilament subunits causes or predisposes to ALS, we used single-strand conformation polymorphism coupled with DNA sequencing to search for mutations in the entirety of the human NF-L, NF-M, and NF-H genes from 100 familial ALS patients known not to carry mutations in superoxide dismutase 1 (SOD1), as well as from 75 sporadic ALS patients. Six polypeptide sequence variants were identified in rod and tail domains of NF-L, NF-M, or NF-H. However, all were found at comparable frequency in DNAs from normal individuals and no variant cosegregated with familial disease. Two deletions found previously in NF-H genes of sporadic ALS patients were not seen in this group of familial or sporadic ALS patients.

Adult↗
PubMed1996

Subunit composition of neurofilaments specifies axonal diameter.

Neurofilaments (NFs), which are composed of NF-L, NF-M, and NF-H, are required for the development of normal axonal caliber, a property that in turn is a critical determinant of axonal conduction velocity. To investigate how each subunit contributes to the radial growth of axons, we used transgenic mice to alter the subunit composition of NFs. Increasing each NF subunit individually inhibits radial axonal growth, while increasing both NF-M and NF-H reduces growth even more severely. An increase in NF-L results in an increased filament number but reduced interfilament distance. Conversely, increasing NF-M, NF-H, or both reduces filament number, but does not alter nearest neighbor interfilament distance. Only a combined increase of NF-L with either NF-M or NF-H promotes radial axonal growth. These results demonstrate that both NF-M and NF-H play complementary roles with NF-L in determining normal axonal calibers.

Animals↗
PubMed1996

Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport.

To examine the mechanism through which neurofilaments regulate the caliber of myelinated axons and to test how aberrant accumulations of neurofilaments cause motor neuron disease, mice have been constructed that express wild-type mouse NF-H up to 4.5 times the normal level. Small increases in NF-H expression lead to increased total neurofilament content and larger myelinated axons, whereas larger increases in NF-H decrease total neurofilament content and strongly inhibit radial growth. Increasing NF-H expression selectively slow neurofilament transport into and along axons, resulting in severe perikaryal accumulation of neurofilaments and proximal axonal swellings in motor neurons. Unlike the situation in transgenic mice expressing modest levels of human NF-H (Cote, F., J.F. Collard, and J.P. Julien. 1993. Cell. 73:35-46), even 4.5 times the normal level of wild-type mouse NF-H does not result in any overt phenotype or enhanced motor neuron degeneration or loss. Rather, motor neurons are extraordinarily tolerant of wild-type murine NF-H, whereas wild-type human NF-H, which differs from the mouse homolog at > 160 residue positions, mediates motor neuron disease in mice by acting as an aberrant, mutant subunit.

Animals↗
PubMed1996

Mechanisms of selective motor neuron death in ALS: insights from transgenic mouse models of motor neuron disease.

Concerning the mechanism(s) of disease underlying amyotrophic lateral sclerosis (ALS), transgenic mouse models have provided (i) a detailed look at the pathogenic progression of disease, (ii) a tool for testing hypotheses concerning the mechanism of neuronal death, and (iii) a host appropriate for testing therapeutic strategies. Thus far, these efforts have proved that mutation in a neurofilament subunit can cause progressive disease displaying both selective motor neuron death and aberrant neurofilament accumulation similar to that reported in human disease. Additional mice expressing point mutations in the cytoplasmic enzyme superoxide dismutase (SOD1), the only known cause of ALS, have proved that disease arises from a toxic property of the mutant enzyme rather than loss of enzymatic activity.

Amyotrophic Lateral Sclerosis↗
PubMed1996

Neuronal intermediate filaments.

Neurofilaments (NFs) are the most abundant structural components in large-diameter myelinated axons. Assembled as obligate heteropolymers requiring NF-L and substoichiometric amounts of NF-M and/or NF-H, NF investment into axons is essential for establishment of axonal caliber, itself a key determinant of conduction velocity. Use of transgenic mice to increase axonal accumulation of NFs or to express mutant NFs subunits has proven that aberrant organization or assembly of NFs is sufficient to cause disease arising from selective dysfunction and degeneration of motor neurons. Because aberrant accumulation of NFs is a common pathology in a series of motor neuron diseases-including amyotrophic lateral sclerosis-NF misaccumulation, and the resultant disruption in axonal transport, is probably a key intermediate in the pathogenesis of these diseases.

Animals↗
PubMed1996

Mechanisms of selective motor neuron death in transgenic mouse models of motor neuron disease.

To examine the mechanism(s) of disease underlying ALS, transgenic mouse models have been constructed that express aberrant neurofilaments or mutations in the abundant, cytoplasmic enzyme superoxide dismutase 1 (SOD1). In addition to progressive weakness arising from selective motor neuron death, mice expressing a modest level of a point mutant in neurofilament subunit NF-L show most of the pathologic hallmarks observed in familial and sporadic ALS, including perikaryal proximal axonal swellings, axonal degeneration, and severe skeletal muscle atrophy. Additional mice expressing familial ALS-linked mutations in the cytoplasmic enzyme SOD1, the only proven cause of ALS and which accounts for approximately 20% of familial disease, have demonstrated that at least one mutation causes disease through acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity. These animals not only provide a detailed look at the pathogenic progression of disease, but also represent a tool for testing hypotheses concerning the specific mechanism(s) of neuronal death and for testing therapeutic strategies.

Amyotrophic Lateral Sclerosis↗
PubMed1996

The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A.

The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

Anaphase↗
PubMed1995

Superoxide dismutase 1 subunits with mutations linked to familial amyotrophic lateral sclerosis do not affect wild-type subunit function.

Mutations in superoxide dismutase 1 (SOD1) have been linked to familial amyotrophic lateral sclerosis, a dominantly inherited motor neuron disorder of midlife. Because SOD1 is a homodimeric enzyme, dimerization of mutant and wild-type SOD1 subunits could dominantly alter the activity, stability, or localization of wild-type SOD1 subunits. To explore these possibilities, we used transient and stable gene transfection to express high levels of either of two mutant human SOD1 subunits in the presence of limited levels of wild-type mouse and/or human SOD1 subunits. Although both mutant subunits displayed diminished half-lives and free radical scavenging activities, their presence caused no change in the half-life or activity of wild-type SOD1 subunits. Our data indicate that mutant subunits do not dominantly affect the function of wild-type SOD1 subunits. These findings, together with observations that many mutant SOD1 subunits retain significant stability and activity, suggest that motor neuron damage in familial amyotrophic lateral sclerosis is caused by the acquisition of injurious properties by mutant SOD1 subunits.

Adult↗
PubMed1995

Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons.

Mutation in superoxide dismutase 1 (SOD1), a Cu/Zn enzyme that removes oxygen radicals and protects against oxidative injury, has been implicated in some cases of familial amyotrophic lateral sclerosis (FALS). As a first approach to examining the mechanism(s) through which these mutations cause specific degeneration of motor neurons, we have used immunocytochemistry to identify the distribution of SOD1 in populations of cells in the peripheral and central nervous systems. In the spinal cord, intense SOD1 immunoreactivity was present in motor neurons, interneurons, and substantia gelatinosa. In motor neurons, SOD1 immunoreactivity was abundant in perikarya, dendrites, and axons; most of this activity appeared to be free in the cytoplasm, although a portion was associated with membranous vesicles, presumably peroxisomes. Since a variety of central nervous system neurons, including pyramidal cells in cerebral cortex and neurons of the CA3 and CA4 sectors of the hippocampus, showed high immunoreactivity but are unaffected in ALS, the apparent abundance of SOD1 does not predict vulnerability of neurons to mutations in SOD1. Rather, SOD1 accumulates in many neuronal populations but is particularly abundant in motor neurons. Consistent with recent studies of FALS-linked SOD1 mutations in vitro and in transgenic mice, our findings offer further support for the view that the mutations confer a gain of adverse function. In this view, high, rather than limiting, levels of SOD1 may place motor neurons selectively at risk in FALS.

Amino Acid Sequence↗
PubMed1995

An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis. Four lines of mice accumulating one of these mutant proteins (G37R) develop severe, progressive motor neuron disease. At lower levels of mutant accumulation, pathology is restricted to lower motor neurons, whereas higher levels cause more severe abnormalities and affect a variety of other neuronal populations. The most obvious cellular abnormality is the presence in axons and dendrites of membrane-bounded vacuoles, which appear to be derived from degenerating mitochondria. Since multiple lines of mice expressing wild-type human SOD1 at similar and higher levels do not show disease, the disease in mice expressing the G37R mutant SOD1 must arise from the acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Increasing neurofilament subunit NF-M expression reduces axonal NF-H, inhibits radial growth, and results in neurofilamentous accumulation in motor neurons.

The carboxy-terminal tail domains of neurofilament subunits neurofilament NF-M and NF-H have been postulated to be responsible for the modulation of axonal caliber. To test how subunit composition affects caliber, transgenic mice were generated to increase axonal NF-M. Total neurofilament subunit content in motor and sensory axons remained essentially unchanged, but increases in NF-M were offset by proportionate decreases in both NF-H and axonal cross-sectional area. Increase in NF-M did not affect the level of phosphorylation of NF-H. This indicates that (a) in vivo NF-H and NF-M compete either for coassembly with a limiting amount of NF-L or as substrates for axonal transport, and (b) NF-H abundance is a primary determinant of axonal caliber. Despite inhibition of radial growth, increase in NF-M and reduction in axonal NF-H did not affect nearest neighbor spacing between neurofilaments, indicating that cross-bridging between nearest neighbors does not play a crucial role in radial growth. Increase in NF-M did not result in an overt phenotype or neuronal loss, although filamentous swellings in perikarya and proximal axons of motor neurons were frequently found.

Amino Acid Sequence↗
PubMed1994

Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity.

Familial amyotrophic lateral sclerosis (FALS) has been linked to mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1). Assay by transient expression in primate cells of six FALS mutant enzymes revealed a continuum of enzymatic activity bounded by the enzyme carrying the mutation Gly-85-->Arg, which was inactive, and mutant enzyme G37R carrying the Gly-37-->Arg change, which retained full specific activity but displayed a 2-fold reduction in polypeptide stability. The G37R mutant displayed similar properties in transformed lymphocytes from an individual heterozygous for the G37R and wild-type SOD1 genes; heterodimeric enzymes composed of mutant and wild-type subunits were detected, but there was no measurable diminution in the stability and activity of the wild-type subunits. Thus, for mutants such as G37R, either surprisingly modest losses in activity (involving only the mutant subunit) can yield motor neuron death, or alternatively, mutant SOD1 may acquire properties that injure motor neurons by one or more mechanisms unrelated to the metabolism of oxygen radicals.

Amino Acid Sequence↗