Biomedical subjects

J Eyer

Publications and source records attributed to J Eyer.

At least 19 recordsLinked to original sources

PubMed2005

Neurofilament high molecular weight-green fluorescent protein fusion is normally expressed in neurons and transported in axons: a neuronal marker to investigate the biology of neurofilaments.

The carboxy-terminal side arm of the neurofilament high subunit consists of a highly phosphorylated domain and a negatively charged region. Multiple evidences suggested that these domains are essential for the axonal phosphorylation and transport of neurofilaments and play a role in their abnormal accumulation following chemical intoxication or during neurodegenerative disorders such as amyotrophic lateral sclerosis. In order to investigate the consequences of altering this side arm of neurofilament high subunit we used a fusion protein (neurofilament high subunit-green fluorescent protein) between the mouse neurofilament high subunit missing a major part of the C-terminal domain and the reporter green fluorescent protein. In cell culture and in transgenic mice this fusion protein co-assembles and co-distributes with the endogenous intermediate filament network. Conditions known to disturb the cytoskeleton were also found to alter the distribution of the fusion protein in cell cultures. In transgenic mice the expression of the transgene evaluated by its fluorescent properties was found to be restricted to neurons, where the neurofilament high subunit-green fluorescent protein fusion protein is axonally transported. Biochemical approaches showed that the fusion protein is phosphorylated and co-purified with neurofilaments. Despite the presence of such an neurofilament high subunit-green fluorescent protein fusion protein, the axonal cytoskeletal density and the axonal caliber were not altered. Together these data show that removal of this portion of neurofilament high subunit does not affect the capacity of neurofilament high subunit to assemble and to be transported into axons, suggesting that this sequence is involved in another function. Moreover, the fluorescent properties of this fusion protein represent a useful marker.

Animals↗

PubMed2004

The amount of neurofilaments aggregated in the cell body is controlled by their increased sensitivity to trypsin-like proteases.

Neurofilaments are synthesised and assembled in neuronal cell bodies, transported along axons and degraded at the synapse. However, in several pathological situations they aggregate in cell bodies or axons. To investigate their turnover when separated from their normal site of degradation, we used a previously described transgenic model characterised by perikaryal retention of neurofilaments, and compared the basic features of both neurofilament synthesis and degradation with that observed in normal mice. Despite the massive perikaryal aggregates, neurofilament transcript levels were found to be unchanged, whereas the total accumulation of neurofilament proteins was markedly reduced. Neurofilaments isolated from transgenic samples are more sensitive to both trypsin and alpha-chymotrypsin mediated proteolysis. Consistent with their greater in vitro sensitivity, trypsin immunolabeling of cell bodies was stronger in transgenic mice. These results show a novel mechanism to regulate the amount of neurofilaments when they abnormally aggregate.

Animals↗

PubMed2003

Characterization of NFH-LacZ transgenic mice with the SHIRPA primary screening battery and tests of motor coordination, exploratory activity, and spatial learning.

NFH-LacZ transgenic mice express a fusion protein between a truncated form of the endogenous neurofilament of heavy molecular weight and the complete E. coli beta-galactosidase. NFH-LacZ transgenic mice could be distinguished from controls in the SHIRPA neurological battery by the appearance of action tremor and hindlimb clasping and a lower body weight. Despite normal exploratory activity and spatial learning, NFH-LacZ transgenic mice were deficient in stationary beam, coat-hanger, and rotorod tests of motor coordination. These results are concordant with neuropathological findings in spinal motoneurons and the cerebellum and indicate that despite the absence of paralysis, these transgenic mice may serve as an experimental model of the early stage of amyotrophic lateral sclerosis.

Journal Article↗

PubMed2003

Stable tubule only polypeptides (STOP) proteins co-aggregate with spheroid neurofilaments in amyotrophic lateral sclerosis.

A major cytopathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of axonal spheroids containing abnormally accumulated neurofilaments. The mechanism of their formation, their contribution to the disease, and the possibility of other co-aggregated components are still enigmatic. Here we analyze the composition of such lesions with special reference to stable tubule only polypeptide (STOP), a protein responsible for microtubule cold stabilization. In normal human brain and spinal cord, the distribution of STOP proteins is uniform between the cytoplasm and neurites of neurons. However, all the neurofilament-rich spheroids present in the tissues of affected patients are intensely labeled with 3 different anti-STOP antibodies. Moreover, when neurofilaments and microtubules are isolated from spinal cord and brain, STOP proteins are systematically co-purified with neurofilaments. By SDS-PAGE analysis, no alteration of the migration profile of STOP proteins is observed in pathological samples. Other microtubular proteins, like tubulin or kinesin, are inconstantly present in spheroids, suggesting that a microtubule destabilizing process may be involved in the pathogenesis of ALS. These results indicate that the selective co-aggregation of neurofilament and STOP proteins represent a new cytopathological marker for spheroids.

Adult↗

PubMed2002

NFH-LacZ transgenic mice: regional brain activity of cytochrome oxidase.

Expression of the NFH-LacZ fusion protein in transgenic mice causes an early accumulation of neurofilament proteins in the cell bodies of neurons, as well as a reduction of motor neuron axonal caliber and Purkinje cell number in the cerebellum. Young (3 month old) and older (12-20 months) NFH-LacZ transgenic mice were compared to normal controls for regional brain metabolism, as assessed by cytochrome oxidase (CO) activity. Irrespective of age, CO activity was reduced in three cerebellar-related regions of NFH-LacZ transgenic mice: (1) the lateral reticular nucleus, (2) the parvicellular red nucleus, and (3) the superior colliculus, possibly as a secondary consequence of cerebellar Purkinje cell histopathology. Aged NFH-LacZ mice had lower CO activity relative to either age-matched controls or young transgenic mice in the following regions: the motor nucleus of the vagus nerve, the trapezoid nucleus, the subiculum, the motor cortex, the superior olive, and the lateral dorsal thalamus. These results indicate regional and age-selective deficits of brain metabolism in a transgenic model with neurofilament maldistribution.

Age Factors↗

PubMed2002

Sensorimotor functions in transgenic mice expressing the neurofilament/heavy-LacZ fusion protein on two genetic backgrounds.

NFH-LacZ transgenic mice are characterized by expression of a non-endogenous fusion protein between a truncated form of mouse NFH (neurofilament of heavy molecular weight) and the complete Escherichia coli beta-galactosidase protein. These transgenic mice were compared to their respective controls on two background strains (C3H and FVB) in several sensorimotor tests. NFH-LacZ mice were deficient in tests requiring balance and equilibrium in a manner generally independent of genetic background. In particular, NFH-LacZ mice fell more quickly than controls from two stationary beams and had fewer rears in an open-field. The transgenic mice were also impaired during the initial trials of sensorimotor learning on the rotorod. We conclude that despite the absence of overt signs of sensorimotor weakness in their home cage, the disruption of the NFH gene, causing neurofilament accumulations in the cell body and diminished axonal calibers of motoneurons, is sufficient to cause motor deficits that resemble the early stages of amyotrophic lateral sclerosis.

Animals↗

PubMed2001

Neurofilament cytoskeleton disruption does not modify accumulation of trophic factor mRNA.

Previously we described a transgenic mouse model in which neurofilaments are sequestered in neuronal cell bodies and withheld from the axonal compartment. This model and other transgenic models with disrupted neurofilaments are used widely to investigate the role of the neurofilament cytoskeleton in normal neurons and in inherited or acquired diseases. To interpret such studies, it is important to establish whether the maldistribution of neurofilaments has major secondary consequences on the cell biology of the affected neurons. Notably, multiple perturbations of the nervous system simultaneously affect both the neuronal cytoskeleton and neurotrophin expression. To determine whether the expression of neurotrophic factors or their receptors is perturbed by a primary disruption in neurofilaments, we compared the accumulated mRNA levels for ciliary neuroptrophic factor (CNTF), nerve growth factor, neurotrophin 3, and the alpha CNTF receptor in mature transgenic mice and their littermate controls. Consistently with the prolonged survival of neurons expressing atypical or abnormally distributed neurofilaments, no obvious changes were observed for any of the mRNA species examined.

Animals↗

PubMed2001

Neurofilaments are nonessential to the pathogenesis of toxicant-induced axonal degeneration.

Axonal neurofilament (NF) accumulations occur before development of symptoms and before other pathological changes among idiopathic neurodegenerative diseases and toxic neuropathies, suggesting a cause-effect relationship. The dependence of symptoms and axonal degeneration on neurofilament accumulation has been tested here in a transgenic mouse model (Eyer and Peterson, 1994) lacking axonal NFs and using two prototypic toxicant models. Chronic acrylamide (ACR) or 2,5-hexanedione exposure resulted in progressive and cumulative increases in sensorimotor deficits. Neurobehavioral tests demonstrated similar expression of neurotoxicity in transgenic (T) mice and their nontransgenic (NT) littermates (containing normal numbers of axonal NFs). Axonal lesions were frequently observed after exposure to either toxicant. Quantitation of ACR-induced lesions demonstrated the distal location of pathology and equal susceptibility of T and NT axons. We conclude that axonal NFs have no effect on neurotoxicity and the pattern of pathology in these mammalian toxic neuropathies. These results also suggest that the role of neurofilament accumulation in the pathogenesis of neurodegenerative diseases requires careful evaluation.

Acrylamide↗

PubMed2000

Neurofilaments are non-essential elements of toxicant-induced reductions in fast axonal transport: pulse labeling in CNS neurons.

Acrylamide (ACR) and g-diketones (g-DK) produce distal sensory-motor neuropathy in a variety of species, including humans. The specific molecular site and mechanism of toxicant action leading to specific morphological and behavioral abnormalities requires definition. The relative roles of fast anterograde axonal transport and neurofilaments (NF) are investigated using optic nerves of mice, with and without axonal neurofilaments. Segmental analysis, following pulse labeling with 3H-leucine into the vitreous body, was used to detect changes in fast anterograde transport in the optic nerve and tract. Single injections of ACR significantly reduced the quantity of radiolabeled proteins transported in both transgenic (lacking NF) and non-transgenic (containing NF) mice by 68.4% and 46.2%, respectively. Similarly, single injections of 2,5-hexanedione (2,5-HD) reduced the quantity of radiolabeled transport in transgenic and non-transgenic mice by 55.2% and 47.1%, respectively. Equimolar doses of propionamide and 3,4-hexanedione (non-neurotoxic analogues of ACR and 2,5-HD, respectively) produced no changes in the quantity or apparent rate of optic nerve transport. Additionally, no differences in quantity or apparent rate of transport between transgenic and non-transgenic animals were observed under control or experimental conditions. Therefore, ACR and 2,5-HD reduce the quantity of fast anterograde axonal transport in mouse CNS axons in a comparable amount to previously reported reductions in rat PNS axons. The absence of axonal neurofilaments had no effect on normal fast transport. Furthermore, the presence or absence of neurofilaments did not alter the effect of these toxicants on fast axonal transport. We conclude that toxicant-induced reductions in fast axonal transport are unrelated to ACR and g-diketone effects on NF or their accumulation.

Acrylamide↗

PubMed1999

Axonal neurofilaments are nonessential elements of toxicant-induced reductions in fast axonal transport: video-enhanced differential interference microscopy in peripheral nervous system axons.

Neurofilament modification and accumulation, occurring in toxicant-induced neuropathies, has been proposed to compromise fast axonal transport and contribute to neurological symptoms or pathology. The current study compares the effects of the neurotoxicants acrylamide (ACR) and 2,5-hexanedione (2,5-HD) on the quantity of fast, bidirectional vesicular traffic within isolated mouse sciatic nerve axons from transgenic mice lacking axonal neurofilaments (Eyer and Peterson, Neuron 12, 1-20, 1994) and nontransgenic littermates possessing neurofilaments. Fast anterograde and retrograde membrane bound organelle (MBO) traffic was quantitated within axons, before and after toxicant exposure, using video-enhanced differential interference contrast (AVEC-DIC) microscopy. Addition of 0.7 mM ACR to the buffer bathing the nerve produced a time-dependent reduction in bidirectional transport with a similar time to onset and magnitude in both transgenic and nontransgenic mice. 2,5-HD (4 mM) exposure reduced bidirectional vesicle traffic by a similar amount in both transgenic and nontransgenic animals. The time to onset of the transport reduction was less and the magnitude of the reduction was greater with 2,5-HD compared to ACR. A single 10-min exposure to ACR or 2,5-HD produced a similar reduction in transport to that produced by prolonged (1 h) exposure. Nonneurotoxic propionamide or 3,4-hexanedione (3,4-HD) produced no changes in bidirectional transport in either transgenic or nontransgenic animals. We conclude that ACR or 2,5-HD produces a rapid, saturable, nonreversible, neurotoxicant-specific reduction in fast bidirectional transport within isolated peripheral nerve axons. These actions are mediated through direct modification of axonal component(s), which are independent of toxicant-induced modifications of, or accumulations of, neurofilaments.

Acrylamide↗

PubMed1999

Motor coordination and spatial orientation are affected by neurofilament maldistribution: correlations with regional brain activity of cytochrome oxidase.

NFH-LacZ transgenic mice are characterized by an early accumulation of the neurofilament cytoskeleton in the cell bodies of neurons with age-associated abnormalities of motor neurons and cerebellar Purkinje cells. In comparison to normal littermate controls, irrespective of age (3 and 12-20 months), NFH-LacZ transgenic mice had a lower number of rears in an open field, deficiencies in some motor-coordination tests, and a higher number of quadrant entries and escape latencies while swimming toward a visible platform. Decreased cytochrome oxidase activity in the lateral reticular nucleus of NFH-LacZ mice was associated with poor performance in two motor coordination tests. Lower metabolic activity in the lateral reticular nucleus may be secondary to previously described cerebellar abnormalities, leading to deficient motor control. The dramatic cytoskeletal perturbation characterizing NFH-LacZ mice affects only selective neuronal populations and results in selective behavioral deficits, which can be correlated with regional brain metabolic activity.

Aging↗

PubMed1998

Pathogenesis of two axonopathies does not require axonal neurofilaments.

Neurofilaments are a major component of the axonal cytoskeleton and their abnormal accumulation is a prominent feature of the cytopathology encountered in several neurodegenerative diseases. Thus, an attractive and widely held model of pathogenesis involves the participation of disrupted neurofilaments as a common toxic intermediate. Here, in direct contrast to this hypothesis, we show that two neurodegenerative disease models in the mouse, dystonia musculorum (dt) and a superoxide dismutase 1 (SOD1)-mediated form of human motor neuron disease (amyotrophic lateral sclerosis, ALS), progress with little or no abatement on a transgenic background in which neurofilaments are withheld from the axonal compartment. By specifically excluding a necessary role for axonal neurofilaments, our observations redefine the components of the pathogenic pathway leading to axon disruption in these two degenerative diseases.

Actin Cytoskeleton↗

PubMed1997

Selective degeneration fo Purkinje cells with Lewy body-like inclusions in aged NFHLACZ transgenic mice.

Transgenic (NFHLacZ) mice expressing a fusion protein composed of a truncated high-molecular-weight mouse neurofilament (NF) protein (NFH) fused to beta-galactosidase (LacZ) develop inclusions in neurons throughout the CNS. These inclusions persist from birth to advanced age and contain massive filamentous aggregates including all three endogenous NF proteins and the NFHLacZ fusion protein. Further, the levels of endogenous NF proteins are selectively reduced in NFHLacZ mice. Because these inclusions resemble NF-rich Lewy bodies (LBs) in Parkinson's disease and LB dementia, we asked whether these lesions compromised the viability of affected neurons during aging. We studied hippocampal CA1 neurons, nearly all of which harbored inclusions (type I) devoid of cellular organelles, and cerebellar Purkinje cells, nearly all of which accumulated inclusions (type II) containing numerous entrapped organelles. Purkinje cells with type II inclusions began to degenerate in the NFHLacZ mice at approximately 1 year of age, and most were eliminated by 18 months of age. In contrast, there was no significant loss of type I inclusion-bearing CA1 neurons with age. These data suggest that the sequestration of cellular organelles in type II inclusions may isolate and impair the function of these organelles, thereby rendering Purkinje cells selectively vulnerable to degeneration with age as in neurodegenerative diseases of the elderly characterized by accumulation of LBs.

Aging↗

PubMed1995

Progressive hyperphosphorylation of neurofilament heavy subunits with aging: possible involvement in the mechanism of neurofilament accumulation.

Abnormal accumulations of phosphorylated neurofilaments occur both in normal senescence and in age-associated neurodegenerative diseases. In the present work, we study the physicochemical properties of neurofilaments isolated from rats of controlled ages. Aging induces in vivo hyperphosphorylation of the heavy neurofilament subunit without affecting in vitro neurofilament phosphorylation by the neurofilament-associated protein kinase. Interactions in vitro between neurofilaments from very old rats occur at higher rate and extent than that of neurofilaments from younger animals. These results support the hypothesis that the abnormal accumulation of neurofilaments observed in nervous tissues from aging mammals results from an altered equilibrium in situ between interconnected and independent neurofilaments.

Aging↗

PubMed1994

Naftidrofuryl, a putative activator of neuron survival, stimulates the expression of neurofilament heavy subunit in cultivated spinal cord neurons from chicken.

The effect of naftidrofuryl, a drug used in ischemia for its vasodilator properties and its protective effect on neuronal survival, was investigated on the maturation of cultured chicken spinal cord neurons, focusing on the presence of proteins specific for the developing neuronal cytoskeleton. Although no influence of naftidrofuryl on the rate of growth of neurites was observed, the drug enhanced the relative amount of the high molecular weight neurofilament subunit without affecting the concentration of a microtubule-associated protein, MAP2. These findings suggest that the effect of naftidrofuryl on cultured spinal cord neurons might involve molecular events directly associated with the induction of a mature cytoskeleton architecture, instead of stimulating undifferentiated neurite growth.

Animals↗

PubMed1994

Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-beta-galactosidase fusion protein.

Interactions between neurofilament side arms may modulate axon caliber. To investigate this hypothesis, we derived transgenic mice expressing a fusion protein in which the carboxyl terminus of the high molecular weight neurofilament protein (NFH) was replaced by beta-galactosidase. The transgene, regulated by NFH sequences, was expressed in projection neurons. However, the fusion protein remained in perikarya precipitating large filamentous aggregates. Axons were not invested with neurofilaments and developed only small calibers. Perikaryal aggregates, with similar structural features, are associated with neurodegenerative diseases, but these mice showed few ill effects and their neurons rarely degenerated. We conclude that an organized neurofilament cytoskeleton is required by axons to achieve large calibers but is not essential for neuronal function or extended survival.

Animals↗

PubMed1992

Age-dependent changes in the ultrastructure and in the molecular composition of rat brain microtubules.

An age-dependent increase of a cathepsin D-like protease activity that preferentially degrades high molecular weight microtubule-associated proteins (MAPs) has been previously described. Microtubules (MT) purified from rat brain of different ages in the presence of several protease inhibitors retained undegraded MAPs through cycles of polymerization, and revealed several age-dependent changes in the relative amounts of MAPs and MT-associated kinases. MAP2 immunoreactivity was found significantly lower in MT preparations from aged animals in contrast with a relative increase of tau molecules. In addition, the phosphorylation of MAP2 by its associated cyclic AMP-dependent protein kinase was also altered, consecutively to the partial loss of the enzyme during polymerization cycles and an age-dependent decrease in the ability of the cyclic nucleotide to stimulate MAP2-bound kinase activity. The evidence of an unusually high packing density of sedimented MT from old rat brains further suggested the modification with aging of the physical structure of the arm-like projections of MAPs, in addition to a lower amount in high molecular weight MAPs. These results support the hypothesis of a selective alteration with aging of the mechanical and regulatory properties of brain MT, consecutive to a change in the composition and/or the structure of MAPs.

Aging↗

PubMed1990

Presence of a new microtubule cold-stabilizing factor in bull sperm dynein preparations.

Brain tubulin polymerized with dynein isolated from bull spermatozoa forms cold-stable microtubules, in contrast with microtubules made of brain tubulin polymerized by brain microtubule-associated proteins (MAPs). The level of cold-stable microtubules depends on the concentration of dynein used. Addition of dynein to cold-unstable microtubules renders these microtubules stable to cold. Although ATP and a non-hydrolysable ATP analogue increase the formation of microtubules made of tubulin and dynein, these nucleotides have no effect on dynein cold-stabilizing properties. The data suggests that a new factor, not involving the dynein ATPase active site and present in bull sperm dynein preparations, confers cold-stability to microtubules.

Adenosine Triphosphate↗